US6207383B1 - Mutations in and genomic structure of HERG—a long QT syndrome gene - Google Patents

Mutations in and genomic structure of HERG—a long QT syndrome gene Download PDF

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US6207383B1
US6207383B1 US09/226,012 US22601299A US6207383B1 US 6207383 B1 US6207383 B1 US 6207383B1 US 22601299 A US22601299 A US 22601299A US 6207383 B1 US6207383 B1 US 6207383B1
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herg
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Mark T. Keating
Igor Splawski
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University of Utah Research Foundation UURF
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Definitions

  • LQT long QT syndrome
  • LQT has been associated with specific genes including HERG, SCN5A, KVLQT1 and KCNE1.
  • LQT may be hereditary and due to specific mutations in the above genes or it may be acquired, e.g., as a result of treatment with drugs given to treat cardiac arrhythmias or of treatment with other types of medications such as antihistamines or antibiotics such as erythromycin.
  • the acquired form of LQT is the more prevalent form of the disorder. It had previously been shown that the HERG gene encodes a K + channel which is involved in the acquired form of LQT.
  • LQT gene carriers manifest prolongation of the QT interval on electrocardiograms, a sign of abnormal cardiac repolarization (Vincent et al., 1992).
  • the clinical features of LQT result from episodic cardiac arrhythmias, specifically torsade de pointes, named for the characteristic undulating nature of the electrocardiogram in this arrhythmia. Torsade de pointes may degenerate into ventricular fibrillation, a particularly lethal arrhythmia.
  • LQT is not a common diagnosis, ventricular arrhythmias are very common; more than 300,000 United States citizens die suddenly every year (Kannel et al., 1987; Willich et al., 1987) and, in many cases, the underlying mechanism may be aberrant cardiac repolarization. LQT, therefore, provides a unique opportunity to study life-threatening cardiac arrhythmias at the molecular level.
  • a more common form of this disorder is called “acquired LQT” and it can be induced by many different factors, particularly treatment with certain medications and reduced serum K + levels (hypokalemia).
  • Autosomal dominant and autosomal recessive forms of the hereditary form of this disorder have been reported.
  • Autosomal recessive LQT also known as Jervell-Lange-Nielsen syndrome
  • Autosomal dominant LQT (Romano-Ward syndrome) is more common, and is not associated with other phenotypic abnormalities.
  • a disorder very similar to inherited LQT can also be acquired, usually as a result of pharmacologic therapy (Schwartz et al., 1975; Zipes, 1987).
  • LQT2 on chromosome 7q35-36 (nine families) and LQT3 on 3p21-24 (three families)
  • LQT3 on 3p21-24 (three families)
  • the genes responsible for LQT at these loci were subsequently identified. These are KVLQT1 (LQT1), HERG (LQT2), and SCN5A (LQT3) (Wang et al., 1996; Curran et al., 1995; Wang et al., 1995; U.S. Pat. No. 5,599,673).
  • KCNE1 (LQT5) was also associated with long QT syndrome (Splawski et al., 1997; Duggal et al., 1998). These genes encode ion channels involved in generation of the cardiac action potential. Mutations can lead to channel dysfunction and delayed myocellular repolarization. Because of regional heterogeneity of channel expression within the myocardium, the aberrant cardiac repolarization creates a substrate for arrhythmia. KVLQT1 and KCNE1 are also expressed in the inner ear (Neyroud et al., 1997; Vetter et al., 1996).
  • Presymptomatic diagnosis of LQT is currently based on prolongation of the QT interval on electrocardiograms.
  • QTc QT interval corrected for heart rate
  • genetic studies have shown that QTc is neither sensitive nor specific (Vincent et al., 1992).
  • the spectrum of QTc intervals for gene carriers and non-carriers overlaps, leading to misclassifications. Non-carriers can have prolonged QTc intervals and be diagnosed as affected.
  • some LQT gene carriers have QTc intervals of ⁇ 0.44 second but are still at increased risk for arrhythmia. Correct presymptomatic diagnosis is important for effective, gene-specific treatment of LQT.
  • HERG human ether a-go-go related gene
  • HERG was localized to human chromosome 7 by PCR analysis of a somatic cell hybrid panel (Warnke and Ganetzky, 1994).
  • the function of the protein encoded by HERG was not known, but it has predicted amino acid sequence homology to potassium channels.
  • HERG was isolated from a hippocampal cDNA library by homology to the Drosophila ether a-go-go gene (eag), which encodes a calcium-modulated potassium channel (Bruggemann et al., 1993).
  • HERG is not the human homolog of eag, however, sharing only ⁇ 50% amino acid sequence homology.
  • the function of HERG was unknown, but it was strongly expressed in the heart and was hypothesized to play an important role in repolarization of cardiac action potentials and was linked to LQT (Curran et al., 1995).
  • Acquired LQT usually results from therapy with medications that block cardiac K + channels (Roden, 1988).
  • the medications most commonly associated with LQT are antiarrhythmic drugs (e.g., quinidine, sotalol) that block the cardiac rapidly-activating delayed rectifier K + current, I Kr , as part of their spectrum of pharmacologic activity.
  • Other drugs may also cause acquired LQT.
  • antihistamines and some antibiotics such as erythromycin.
  • I Kr has been characterized in isolated cardiac myocytes (Balser et al., 1990; Follmer et al., 1992; Sanguinetti and Jurkiewicz, 1990; Shibasaki, 1987; T. Yang et al., 1994), and is known to have an important role in initiating repolarization of action potentials.
  • HERG encodes a K + channel with biophysical characteristics nearly identical to I Kr .
  • the HERG genomic structure is defined showing that it comprises 15 exons and spans 55 kilobases. Primer pairs are presented which allow analysis of all 15 exons for mutations which may be associated with long QT syndrome. Many new mutations in HERG associated with long QT syndrome are also presented.
  • FIGS. 1A-D Currents elicited by depolarizing voltage steps in Xenopus oocytes injected with HERG cRNA.
  • FIG. 1 A Currents activated by 4 sec pulses, applied in 10 mV increments from ⁇ 50 to ⁇ 10 mV. Current during the pulse progressively increased with voltage, as did tail current upon return to the holding potential. Holding potential was ⁇ 70 mV. The inset illustrates the voltage pulse protocol.
  • FIG. 1 B Currents activated with test pulses of 0 to +40 mV, applied in 10 mV increments. Current magnitude during the pulse progressively decreased with voltage, whereas the tail current saturated at +10 mV. Note that currents do not exhibit slow inactivation.
  • FIG. 1 A Currents activated by 4 sec pulses, applied in 10 mV increments from ⁇ 50 to ⁇ 10 mV. Current during the pulse progressively increased with voltage, as did tail current upon return to the holding potential. Holding potential was ⁇ 70 mV
  • FIGS. 2A-D Kinetics of HERG current activation and deactivation.
  • FIG. 2 B Deactivation of HERG currents. Current was activated with 1.6 sec pulses to +20 mV, followed by return to test potentials ranging from ⁇ 40 to ⁇ 100 mV (10 mV steps).
  • FIGS. 3A-C Reversal potential of HERG current varies with [K + ] e as expected for a K + -selective channel.
  • the estimated reversal potential of tail currents was ⁇ 97 mV. Currents were not leak subtracted.
  • the dotted line is the relationship predicted by the Nernst equation for a perfectly K + -selective channel.
  • the relative permeability of Na + to K + (r) determined from this fit was 0.007.
  • FIGS. 4A-E Activation of HERG current by extracellular K + .
  • FIGS. 4 A-C Currents elicited by 4 sec pulses to test potentials ranging from ⁇ 50 to +20 mV in an oocyte bathed in modified ND96 solution containing 10 mM KCl (A), 2 mM KCl (B), or 5 min after switching to ND96 solution with no added KCl (C).
  • FIG. 4 D Current-voltage relationship for currents shown in panels A-C.
  • FIGS. 5A-D HERG rectification results from rapid inactivation.
  • FIG. 5 B Time constants for recovery from fast inactivation determined from fits of tail currents as described above.
  • FIG. 5 C Fully-activated HERG I-V relationship.
  • FIG. 5 D Voltage-dependence of rapid inactivation of HERG current.
  • the rectification factor, R, at each potential was calculated using current amplitudes plotted in panel (C):
  • the data were fit with a Boltzmann equation: 1/(1+exp[(E rev ⁇ V 1 ⁇ 2 )/k]). The value of V 1 ⁇ 2 was ⁇ 49 mV and the slope factor (k) was +28 mV.
  • FIGS. 6A-D HERG current is blocked by La 3+ .
  • FIG. 6 A Control currents activated by 4 sec pulses to potentials ranging from ⁇ 50 to +50 mV. Currents were not leak subtracted.
  • FIG. 6 B Currents elicited with the same pulse protocol after exposure of oocyte to 10 ⁇ M LaCl 3 .
  • FIG. 6 C I-V relationship of HERG currents measured at the end of 4 sec test pulses.
  • FIG. 6 D Isochronal activation curves were determined from plots of tail current amplitudes as a function of test potential. Data were fitted to a Boltzmann function to obtain the smooth isochronal activation curve. La 3+ shifted the half-point of activation from ⁇ 16 mV to +23 mV.
  • FIG. 7 Physical map and exon organization of HERG.
  • the genomic region of HERG encompasses approximately 55 kilobases.
  • the overlapping cosmid clones containing the entire HERG transcript sequence are shown.
  • the location of HERG exons relative to genomic clones is indicated. Sizes of exons and distances are not drawn to scale.
  • FIGS. 8A-B Genomic organization of HERG coding and 5′ and 3′ untranslated sequences. Positions of introns are indicated with arrowheads. The six putative membrane-spanning segments (S1 to S6) and the putative pore (Pore) and cyclic nucleotide binding (cNBD) regions are underlined. The asterisk marks the stop codon.
  • the nucleic acid and protein of FIGS. 8A-B are SEQ ID NO:3 and SEQ ID NO:4, respectively.
  • FIGS. 9A-E Pedigree structure and genotypic analyses of five new LQT families. Individuals showing the characteristic features of LQT, including prolongation of the QT interval and history of syncope, seizures or aborted sudden death, are indicated by filled circles (females) or squares (males). Unaffected individuals are indicated by empty circles or squares. Individuals with an equivocal phenotype, or for whom phenotypic data are unavailable, are stippled. Circles or squares with a slash denote deceased individuals. Haplotypes for polymorphic markers linked to LQT2 are shown under each individual.
  • markers include (centromere to telomere) D7S505, D7S636, HERG 5-11, HERG 3-8, D7S483 (Gyapay et al., 1994; Wang et al., 1995).
  • Haplotypes cosegregating with the disease phenotype are indicated by a box. Recombination events are indicated with a horizontal black line. Informed consent was obtained from all individuals, or their guardians, in accordance with local institutional review board guidelines. Haplotype analyses indicate that the LQT phenotype in these kindreds is linked to markers on chromosome 7q35-36.
  • FIGS. 10A-C HERG intragenic deletions associated with LQT in two families. Pedigree structure of K2287 (FIG. 10 A), results of PCR amplification using primer pair 1-9 (FIG. 10 A), results of DNA sequencing of normal and mutant K2287 HERG genes (FIG. 10 B), and the effect of the deletion on predicted structure of HERG protein (FIG. 10C) are shown. Note that an aberrant fragment of 143 bp is observed in affected members of this kindred, indicating the presence of a disease-associated intragenic deletion. DNA sequence of normal and aberrant PCR products defines a 27 bp deletion ( ⁇ I500-F508). This mutation causes an in-frame deletion of 9 amino acids in the third membrane spanning domain (S3). Deleted sequences are indicated.
  • FIGS. 11A-C Pedigree structure of K2595 is shown (FIG. 11 A). Deceased individuals are indicated by a slash. The result of SSCP analyses using primer pair 1-9 are shown beneath each individual (FIG. 11 A). Note that an aberrant SSCP conformer cosegregates with the disease in this family.
  • DNA sequence shows a single base-pair deletion ( ⁇ 1261) (FIG. 11 B). This deletion results in a frameshift followed by a stop codon 12 amino acids downstream (FIG. 11 C). The deleted nucleotide is indicated with an arrow.
  • FIGS. 12A-I HERG point mutations identified in three LQT kindreds.
  • Pedigree structure of K1956 (FIG. 12 A), K2596 (FIG. 12C) and K2015 (FIG. 12E) are shown.
  • the results of SSCP analyses with primer pair 5-11 (K1956) (FIG. 12 B), primer pair 1-9 (K2596) (FIG. 12D) and primer pair 4-12 (K2015) (FIG. 12F) are shown.
  • Aberrant SSCP conformers cosegregate with the disease in each kindred.
  • DNA sequence analyses of the normal and aberrant conformers reveals a C to T substitution at position 1682 in K1956.
  • FIGS. 13A-E HERG missense mutations associated with LQT. Results from SSCP analyses and the mutation effect on amino acid sequence are shown below each pedigree. Note that aberrant SSCP conformers (indicated by an arrow) cosegregate with the disease phenotype.
  • FIGS. 14A-C De novo mutation of HERG in a sporadic case of LQT.
  • Pedigree structure of K2269 (FIG. 14A) and SSCP analyses (primer pair 14-16) (FIG. 14A) showing an aberrant conformer in a sporadic case of LQT.
  • DNA sequence analyses identified a G to A substitution at position 1882 of the cDNA sequence (C to T substitution on the antisense-strand is shown) (FIG. 14 B). Note that this mutation results in the substitution of a serine for a highly conserved glycine residue at codon 628 (G628S) (FIG. 14 C). This amino acid sequence is known to be critical for potassium ion selectivity.
  • FIG. 15 Northern blot analysis of HERG mRNA showing strong expression in the heart.
  • a Northern blot (Clonetech, poly A ⁇ RNA, 2 mg/lane) was probed using an HERG cDNA containing nucleotides 679 to 2239 of the coding sequence. Two cardiac mRNAs of ⁇ 4.1 and 4.4 kb are indicated. Background in mRNA extracted from lung was high, but no specific bands were identified.
  • SEQ ID NO:1 is the nucleic acid coding region only of HERG cDNA.
  • SEQ ID NO:2 is the HERG protein encoded by SEQ ID NO:1.
  • SEQ ID NO:3 is the nucleic acid of HERG cDNA and includes the complete coding region as well as some 5′ and 3′ untranslated regions.
  • SEQ ID NO:4 is the HERG protein encoded by SEQ ID NO:3.
  • SEQ ID Nos:5 and 6 are hypothetical nucleic acids used to demonstrate the calculation of percent homology.
  • SEQ ID NOs:7 and 8 are primers for amplifying the 3′ UTR of HERG.
  • SEQ ID NOs:9-25 are primer pairs for SSCP analysis (Table 3).
  • SEQ ID NOs:26-55 are the intron/exon boundaries of HERG (Table 4).
  • SEQ ID Nos:56-95 are primers to amplify HERG exons (Table 5).
  • SEQ ID Nos:96-97 show the deletion of K2287 (FIG. 10 C).
  • SEQ ID Nos:98-101 show the effect of the deletion in K2595 (FIG. 11 C).
  • SEQ ID NOs:102-116 are a comparison of regions of HERG from humans, mouse, rat and Drosophila (FIGS. 12G-H and 14 C).
  • the present invention is directed to the genomic structure of HERG and to newly found mutations in HERG associated with LQT.
  • the present invention is further directed to methods of screening humans for the presence of HERG gene variants which cause LQT. Since LQT can now be detected earlier (i.e., before symptoms appear) and more definitively, better treatment options will be available in those individuals identified as having hereditary LQT.
  • the present invention provides methods of screening the HERG gene to identify mutations. Such methods may further comprise the step of amplifying a portion of the HERG gene, and may further include a step of providing a set of polynucleotides which are primers for amplification of said portion of the HERG gene. The method is useful for identifying mutations for use in either diagnosis of LQT or prognosis of LQT.
  • Long QT syndrome is an inherited or an acquired disorder that causes sudden death from cardiac arrhythmias, specifically torsade de pointes and ventricular fibrillation.
  • LQT was previously mapped to four loci: KVLQT1 on chromosome 11p15.5, HERG on 7q35-36, SCN5A on 3p21-24 and KCNE1 on chromosome 21q22.1-22.2.
  • LQT susceptibility alleles will co-segregate with the disease in large kindreds. They will also be present at a much higher frequency in non-kindred individuals with LQT than in individuals in the general population.
  • the key is to find mutations which are serious enough to cause obvious disruption to the normal function of the gene product. These mutations can take a number of forms. The most severe forms would be frame shift mutations or large deletions which would cause the gene to code for an abnormal protein or one which would significantly alter protein expression.
  • Less severe disruptive mutations would include small in-frame deletions and nonconservative base pair substitutions which would have a significant effect on the protein produced, such as changes to or from a cysteine residue, from a basic to an acidic amino acid or vice versa, from a hydrophobic to hydrophilic amino acid or vice versa, or other mutations which would affect secondary or tertiary protein structure. Silent mutations or those resulting in conservative amino acid substitutions would not generally be expected to disrupt protein function.
  • alteration of the wild-type HERG gene is detected.
  • the method can be performed by detecting the wild-type HERG gene and confirming the lack of a cause of LQT as a result of a mutation at this locus.
  • “Alteration of a wild-type gene” encompasses all forms of mutations including deletions, insertions and point mutations in the coding and noncoding regions. Deletions may be of the entire gene or of only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certain tissues and are not inherited in the germline.
  • Germline mutations can be found in any of a body's tissues and are inherited. Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the HERG gene product, or to a decrease in mRNA stability or translation efficiency.
  • the presence of hereditary LQT may be ascertained by testing any tissue of a human for mutations of the HERG gene. For example, a person who has inherited a germline HERG mutation would be prone to develop LQT. This can be determined by testing DNA from any tissue of the person's body. Most simply, blood can be drawn and DNA extracted from the cells of the blood. In addition, prenatal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic cells for mutations of the HERG gene. Alteration of a wild-type HERG allele, whether, for example, by point mutation or deletion, can be detected by any of the means discussed herein.
  • SSCP single-stranded conformation polymorphism assay
  • CDGE clamped denaturing gel electrophoresis
  • HA heteroduplex analysis
  • CMC chemical mismatch cleavage
  • a rapid preliminary analysis to detect polymorphisms in DNA sequences can be performed by looking at a series of Southern blots of DNA cut with one or more restriction enzymes, preferably with a large number of restriction enzymes. Each blot contains a series of normal individuals and a series of LQT cases. Southern blots displaying hybridizing fragments (differing in length from control DNA when probed with sequences near or including the HERG locus) indicate a possible mutation. If restriction enzymes which produce very large restriction fragments are used, then pulsed field gel electrophoresis (PFGE) is employed.
  • PFGE pulsed field gel electrophoresis
  • Detection of point mutations may be accomplished by molecular cloning of the HERG alleles and sequencing the alleles using techniques well known in the art. Also, the gene or portions of the gene may be amplified, e.g., by PCR or other amplification technique, and the amplified gene or amplified portions of the gene may be sequenced.
  • coli mutS protein (Modrich, 1991); and 6) allele-specific PCR (Ruano and Kidd, 1989).
  • primers are used which hybridize at their 3′ ends to a particular HERG mutation. If the particular mutation is not present, an amplification product is not observed.
  • Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Application Publication No. 0332435 and in Newton et al., 1989. Insertions and deletions of genes can also be detected by cloning, sequencing and amplification.
  • RFLP restriction fragment length polymorphism
  • Such a method is particularly useful for screening relatives of an affected individual for the presence of the mutation found in that individual.
  • Other techniques for detecting insertions and deletions as known in the art can be used.
  • SSCP detects a band which migrates differentially because the sequence change causes a difference in single-strand, intramolecular base pairing.
  • RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments.
  • DGGE detects differences in migration rates of mutant sequences compared to wild-type sequences, using a denaturing gradient gel.
  • an allele-specific oligonucleotide assay an oligonucleotide is designed which detects a specific sequence, and the assay is performed by detecting the presence or absence of a hybridization signal.
  • the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild-type sequences.
  • Mismatches are hybridized nucleic acid duplexes in which the two strands are not 100% complementary. Lack of total homology may be due to deletions, insertions, inversions or substitutions. Mismatch detection can be used to detect point mutations in the gene or in its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of samples.
  • An example of a mismatch cleavage technique is the RNase protection method. In the practice of the present invention, the method involves the use of a labeled riboprobe which is complementary to the human wild-type HERG gene coding sequence.
  • the riboprobe and either mRNA or DNA isolated from the person are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch. Thus, when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA.
  • the riboprobe need not be the full length of the mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the mRNA or gene, it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches.
  • DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Cotton et al., 1988; Shenk et al., 1975; Novack et al., 1986.
  • mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, e.g., Cariello, 1988.
  • the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridization. Changes in DNA of the HERG gene can also be detected using Southern hybridization, especially if the changes are gross rearrangements, such as deletions and insertions.
  • DNA sequences of the HERG gene which have been amplified by use of PCR may also be screened using allele-specific probes.
  • These probes are nucleic acid oligomers, each of which contains a region of the gene sequence harboring a known mutation. For example, one oligomer may be about 30 nucleotides in length, corresponding to a portion of the gene sequence.
  • PCR amplification products can be screened to identify the presence of a previously identified mutation in the gene.
  • Hybridization of allele-specific probes with amplified HERG sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under high stringency hybridization conditions indicates the presence of the same mutation in the tissue as in the allele-specific probe.
  • the newly developed technique of nucleic acid analysis via microchip technology is also applicable to the present invention.
  • this technique literally thousands of distinct oligonucleotide probes are built up in an array on a silicon chip. Nucleic acid to be analyzed is fluorescently labeled and hybridized to the probes on the chip. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips. Using this technique one can determine the presence of mutations or even sequence the nucleic acid being analyzed or one can measure expression levels of a gene of interest.
  • the method is one of parallel processing of many, even thousands, of probes at once and can tremendously increase the rate of analysis.
  • the most definitive test for mutations in a candidate locus is to directly compare genomic HERG sequences from patients with those from a control population.
  • Mutations from patients falling outside the coding region of HERG can be detected by examining the non-coding regions, such as introns and regulatory sequences near or within the genes.
  • An early indication that mutations in noncoding regions are important may come from Northern blot experiments that reveal messenger RNA molecules of abnormal size or abundance in patients as compared to control individuals.
  • Alteration of HERG mRNA expression can be detected by any techniques known in the art. These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA expression indicates an alteration of the wild-type gene. Alteration of wild-type genes can also be detected by screening for alteration of wild-type HERG protein. For example, monoclonal antibodies immunoreactive with HERG can be used to screen a tissue. Lack of cognate antigen would indicate a mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant gene product. Such immunological assays can be done in any convenient formats known in the art. These include Western blots, inumunohistochemical assays and ELISA assays.
  • Any means for detecting an altered HERG protein can be used to detect alteration of wild-type HERG genes.
  • Functional assays such as protein binding determinations, can be used.
  • assays can be used which detect HERG biochemical function. Finding a mutant HERG gene product indicates alteration of a wild-type HERG gene.
  • Mutant HERG genes or gene products can also be detected in other human body samples, such as serum, stool, urine and sputum.
  • Other human body samples such as serum, stool, urine and sputum.
  • the same techniques discussed above for detection of mutant genes or gene products in tissues can be applied to other body samples. By screening such body samples, a simple early diagnosis can be achieved for hereditary LQT.
  • the primer pairs of the present invention are useful for determination of the nucleotide sequence of a particular HERG allele using PCR.
  • the pairs of single-stranded DNA primers can be annealed to sequences within or surrounding the HERG gene on chromosome 7 in order to prime amplifying DNA synthesis of the gene itself.
  • a complete set of these primers allows synthesis of all of the nucleotides of the gene coding sequences, i.e., the exons.
  • the set of primers preferably allows synthesis of both intron and exon sequences. Allele-specific primers can also be used. Such primers anneal only to particular HERG mutant alleles, and thus will only amplify a product in the presence of the mutant allele as a template.
  • primers may have restriction enzyme site sequences appended to their 5′ ends.
  • all nucleotides of the primers are derived from HERG sequences or sequences adjacent to HERG, except for the few nucleotides necessary to form a restriction enzyme site.
  • the primers themselves can be synthesized using techniques which are well known in the art. Generally, the primers can be made using oligonucleotide synthesizing machines which are commercially available. Given the cDNA sequence of HERG (Warmke and Ganetzky, 1994), design of particular primers is well within the skill of the art. The present invention adds to this by presenting data on the intron/exon boundaries thereby allowing one to design primers to amplify and sequence all of the exonic regions completely.
  • the nucleic acid probes provided by the present invention are useful for a number of purposes. They can be used in Southern hybridization to genomic DNA and in the RNase protection method for detecting point mutations already discussed above.
  • the probes can be used to detect PCR amplification products. They may also be used to detect mismatches with the HERG gene or mRNA using other techniques.
  • mutant alleles can be initially identified by identifying mutant (altered) proteins, using conventional techniques. The mutant alleles are then sequenced to identify the specific mutation for each allele. The mutations, especially those which lead to an altered function of the protein, are then used for the diagnostic and prognostic methods of the present invention.
  • the present invention also provides methods of treating patients with K + to decrease the chances of developing LQT and/or torsade de pointes.
  • the modulation of HERG by extracellular K + ([K + ] e ) may have physiologic importance.
  • K + accumulates within intracellular clefts (Gintant et al., 1992). This elevation in [K + ] e would increase the contribution of HERG to net repolarizing current.
  • HERG may be even more important, therefore, in modulation of action potential duration at high heart rates, or during the initial phase of ischemia.
  • the present invention employs the following definitions:
  • “Amplification of Polynucleotides” utilizes methods such as the polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR) and amplification methods based on the use of Q-beta replicase. Also useful are strand displacement amplification (SDA), thermophilic SDA, and nucleic acid sequence based amplification (3SR or NASBA). These methods are well known and widely practiced in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); Wu and Wallace, 1989 (for LCR); U.S. Pat. Nos.
  • analyte polynucleotide and “analyte strand” refer to a single- or double-stranded polynucleotide which is suspected of containing a target sequence, and which may be present in a variety of types of samples, including biological samples.
  • Antibodies The present invention also provides polyclonal and/or monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof, which are capable of specifically binding to the HERG polypeptide and fragments thereof or to polynucleotide sequences from the HERG region.
  • the term “antibody” is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities.
  • Polypeptides may be prepared synthetically in a peptide synthesizer and coupled to a carrier molecule (e.g., keyhole limpet hemocyanin) and injected over several months into rabbits. Rabbit sera is tested for immunoreactivity to the HERG polypeptide or fragment.
  • a carrier molecule e.g., keyhole limpet hemocyanin
  • Monoclonal antibodies may be made by injecting mice with the protein polypeptides, fusion proteins or fragments thereof. Monoclonal antibodies will be screened by ELISA and tested for specific immunoreactivity with HERG polypeptide or fragments thereof. See, Harlow and Lane, 1988. These antibodies will be useful in assays as well as pharmaceuticals.
  • antibodies specific for binding may be either polyclonal or monoclonal, and may be produced by in vitro or in vivo techniques well known in the art.
  • an appropriate target immune system typically mouse or rabbit
  • Substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal and by other parameters well known to immunologists. Typical sites for injection are in footpads, intramuscularly, intraperitoneally, or intradermally. Of course, other species may be substituted for mouse or rabbit.
  • Polyclonal antibodies are then purified using techniques known in the art, adjusted for the desired specificity.
  • An immunological response is usually assayed with an immunoassay.
  • immunoassays involve some purification of a source of antigen, for example, that produced by the same cells and in the same fashion as the antigen.
  • a variety of immunoassay methods are well known in the art. See, e.g., Harlow and Lane, 1988, or Goding, 1986.
  • Monoclonal antibodies with affinities of 10 ⁇ 8 M ⁇ 1 or preferably 10 ⁇ 9 to 10 ⁇ 10 M ⁇ 1 or stronger will typically be made by standard procedures as described, e.g., in Harlow and Lane, 1988 or Goding, 1986. Briefly, appropriate animals will be selected and the desired immunization protocol followed. After the appropriate period of time, the spleens of such animals are excised and individual spleen cells fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter, the cells are clonally separated and the supernatants of each clone tested for their production of an appropriate antibody specific for the desired region of the antigen.
  • Suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides, or alternatively, to selection of libraries of antibodies in phage or similar vectors. See Huse et al., 1989.
  • the polypeptides and antibodies of the present invention may be used with or without modification. Frequently, polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. Patents teaching the use of such labels include U.S. Pat. Nos.
  • Binding partner refers to a molecule capable of binding a ligand molecule with high specificity, as for example, an antigen and an antigen-specific antibody or an enzyme and its inhibitor.
  • the specific binding partners must bind with sufficient affinity to immobilize the analyte copy/complementary strand duplex (in the case of polynucleotide hybridization) under the isolation conditions.
  • Specific binding partners are known in the art and include, for example, biotin and avidin or streptavidin, IgG and protein A, the numerous, known receptor-ligand couples, and complementary polynucleotide strands.
  • the partners are normally at least about 15 bases in length, and may be at least 40 bases in length. It is well recognized by those of skill in the art that lengths shorter than 15 (e.g., 8 bases), between 15 and 40, and greater than 40 bases may also be used.
  • the polynucleotides may be composed of DNA, RNA, or synthetic nucleotide analogs. Further binding partners can be identified using, e.g., the two-hybrid yeast screening assay as described herein.
  • a “biological sample” refers to a sample of tissue or fluid suspected of containing an analyte polynucleotide or polypeptide from an individual including, but not limited to, e.g., plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, blood cells, tumors, organs, tissue and samples of in vitro cell culture constituents.
  • Encode A polynucleotide is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof.
  • the anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • nucleic acid e.g., an RNA, DNA or a mixed polymer
  • An “isolated” or “substantially pure” nucleic acid is one which is substantially separated from other cellular components which naturally accompany a native human sequence or protein, e.g., ribosomes, polymerases, many other human genome sequences and proteins.
  • the term embraces a nucleic acid sequence or protein which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
  • HERG Allele refers to normal alleles of the HERG locus as well as alleles of HERG carrying variations that cause LQT.
  • HERG Locus each refer to polynucleotides, all of which are in the HERG region, that are likely to be expressed in normal tissue, certain alleles of which result in LQT.
  • the HERG locus is intended to include coding sequences, intervening sequences and regulatory elements controlling transcription and/or translation.
  • the HERG locus is intended to include all allelic variations of the DNA sequence.
  • nucleic acid when applied to a nucleic acid, refer to a nucleic acid which encodes a human HERG polypeptide, fragment, homolog or variant, including, e.g., protein fusions or deletions.
  • the nucleic acids of the present invention will possess a sequence which is either derived from, or substantially similar to a natural HERG-encoding gene or one having substantial homology with a natural HERG-encoding gene or a portion thereof.
  • the HERG gene or nucleic acid includes normal alleles of the HERG gene, including silent alleles having no effect on the amino acid sequence of the HERG polypeptide as well as alleles leading to amino acid sequence variants of the HERG polypeptide that do not substantially affect its function. These terms also include alleles having one or more mutations which adversely affect the function of the HERG polypeptide.
  • a mutation may be a change in the HERG nucleic acid sequence which produces a deleterious change in the amino acid sequence of the HERG polypeptide, resulting in partial or complete loss of HERG function, or may be a change in the nucleic acid sequence which results in the loss of effective HERG expression or the production of aberrant forms of the HERG polypeptide.
  • the HERG nucleic acid may be that shown in SEQ ID NO:1 (coding region of HERG cDNA) or SEQ ID NO:3 (cDNA including 5′ UTR and 3′ UTR) or it may be an allele as described above or a variant or derivative differing from that shown by a change which is one or more of addition, insertion, deletion and substitution of one or more nucleotides of the sequence shown. Changes to the nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the genetic code.
  • nucleic acid according to the present invention may include a sequence different from the sequence shown in SEQ ID NOs:1 and 3 yet encode a polypeptide with the same amino acid sequence as shown in SEQ ID NOs:2 and 4. That is, nucleic acids of the present invention include sequences which are degenerate as a result of the genetic code.
  • the encoded polypeptide may comprise an amino acid sequence which differs by one or more amino acid residues from the amino acid sequence shown in SEQ ID NOs:2 and 4.
  • Nucleic acid encoding a polypeptide which is an amino acid sequence variant, derivative or allele of the amino acid sequence shown in SEQ ID NOs:2 and 4 is also provided by the present invention.
  • the HERG gene also refers to (a) any DNA sequence that (i) hybridizes to the complement of the DNA sequences that encode the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4 under highly stringent conditions (Ausubel et al., 1992) and (ii) encodes a gene product functionally equivalent to HERG, or (b) any DNA sequence that (i) hybridizes to the complement of the DNA sequences that encode the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4 under less stringent conditions, such as moderately stringent conditions (Ausubel et al., 1992) and (ii) encodes a gene product functionally equivalent to HERG.
  • the invention also includes nucleic acid molecules that are the complements of the sequences described herein.
  • the polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • pendent moieties
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • the present invention provides recombinant nucleic acids comprising all or part of the HERG region.
  • the recombinant construct may be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct may become integrated into the chromosomal DNA of the host cell.
  • a recombinant polynucleotide comprises a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin which, by virtue of its origin or manipulation, 1) is not associated with all or a portion of a polynucleotide with which it is associated in nature; 2) is linked to a polynucleotide other than that to which it is linked in nature; or 3) does not occur in nature.
  • nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as reference to the RNA equivalent, with U substituted for T.
  • nucleic acids comprising sequences otherwise not naturally occurring are provided by this invention.
  • wild-type sequence may be employed, it will often be altered, e.g., by deletion, substitution or insertion.
  • cDNA or genomic libraries of various types may be screened as natural sources of the nucleic acids of the present invention, or such nucleic acids may be provided by amplification of sequences resident in genomic DNA or other natural sources, e.g., by PCR.
  • the choice of cDNA libraries normally corresponds to a tissue source which is abundant in mRNA for the desired proteins. Phage libraries are normally preferred, but other types of libraries may be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences.
  • the DNA sequences used in this invention will usually comprise at least about five codons (15 nucleotides), more usually at least about 7-15 codons, and most preferably, at least about 35 codons. One or more introns may also be present. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with a HERG-encoding sequence. In this context, oligomers of as low as 8 nucleotides, more generally 8-17 nucleotides, can be used for probes, especially in connection with chip technology.
  • nucleic acid manipulation is described generally, for example, in Sambrook et al., 1989 or Ausubel et al., 1992.
  • Reagents useful in applying such techniques such as restriction enzymes and the like, are widely known in the art and commercially available from such vendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega, U.S. Biochemicals, New England Nuclear, and a number of other sources.
  • the recombinant nucleic acid sequences used to produce fusion proteins of the present invention may be derived from natural or synthetic sequences. Many natural gene sequences are obtainable from various cDNA or from genomic libraries using appropriate probes. See, GenBank, National Institutes of Health.
  • a “portion” of the HERG locus or region or allele is defined as having a minimal size of at least about eight nucleotides, or preferably about 15 nucleotides, or more preferably at least about 25 nucleotides, and may have a minimal size of at least about 40 nucleotides. This definition includes all sizes in the range of 8-40 nucleotides as well as greater than 40 nucleotides.
  • this definition includes nucleic acids of 8, 12, 15, 20, 25, 40, 60, 80, 100, 200, 300, 400, 500 nucleotides, or nucleic acids having any number of nucleotides within these ranges of values (e.g., 9, 10, 11, 16, 23, 30, 38, 50, 72, 121, etc., nucleotides), or nucleic acids having more than 500 nucleotides.
  • the present invention includes all novel nucleic acids having at least 8 nucleotides derived from SEQ ID NO:1 or SEQ ID NO:3, its complement or functionally equivalent nucleic acid sequences.
  • the present invention does not include nucleic acids which exist in the prior art. That is, the present invention includes all nucleic acids having at least 8 nucleotides derived from SEQ ID NO:1 or SEQ ID NO:3 with the proviso that it does not include nucleic acids existing in the prior art.
  • HERG protein or “HERG polypeptide” refers to a protein or polypeptide encoded by the HERG locus, variants or fragments thereof.
  • polypeptide refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not refer to, or exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • polypeptides with substituted linkages as well as other modifications known in the art, both naturally and non-naturally occurring.
  • polypeptides will be at least about 50% homologous to the native HERG sequence, preferably in excess of about 90%, and more preferably at least about 95% homologous.
  • proteins encoded by DNA which hybridize under high or low stringency conditions, to HERG-encoding nucleic acids and closely related polypeptides or proteins retrieved by antisera to the HERG protein(s).
  • the HERG polypeptide may be that shown in SEQ ID NO:2 or SEQ ID NO:4 which may be in isolated and/or purified form, free or substantially free of material with which it is naturally associated.
  • the polypeptide may, if produced by expression in a prokaryotic cell or produced synthetically, lack native post-translational processing, such as glycosylation.
  • the present invention is also directed to polypeptides which are sequence variants, alleles or derivatives of the HERG polypeptide.
  • Such polypeptides may have an amino acid sequence which differs from that set forth in SEQ ID NO:2 or SEQ ID NO:4 by one or more of addition, substitution, deletion or insertion of one or more amino acids.
  • Preferred such polypeptides have HERG function.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties.
  • Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • Preferred substitutions are ones which are conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and tyrosine, phenylalanine.
  • Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules or binding sites on proteins interacting with the HERG polypeptide. Since it is the interactive capacity and nature of a protein which defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydrophobic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982).
  • hydrophilicity in conferring interactive biological function of a protein is generally understood in the art (U.S. Pat. No. 4,554,101).
  • hydrophobic index or hydrophilicity in designing polypeptides is further discussed in U.S. Pat. No. 5,691,198.
  • the length of polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.
  • “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • peptide mimetic or mimetic is intended to refer to a substance which has the essential biological activity of the HERG polypeptide.
  • a peptide mimetic may be a peptide-containing molecule that mimics elements of protein secondary structure (Johnson et al., 1993).
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen, enzyme and substrate or scaffolding proteins.
  • a peptide mimetic is designed to permit molecular interactions similar to the natural molecule.
  • a mimetic may not be a peptide at all, but it will retain the essential biological activity of natural HERG polypeptide.
  • Probes Polynucleotide polymorphisms associated with HERG alleles which predispose to LQT are detected by hybridization with a polynucleotide probe which forms a stable hybrid with that of the target sequence, under stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes will be perfectly complementary to the target sequence, high stringency conditions will be used. Hybridization stringency may be lessened if some mismatching is expected, for example, if variants are expected with the result that the probe will not be completely complementary. Conditions are chosen which rule out nonspecific/adventitious bindings, that is, which minimize noise.
  • Probes for HERG alleles may be derived from the sequences of the HERG region, its cDNA, functionally equivalent sequences, or the complements thereof.
  • the probes may be of any suitable length, which span all or a portion of the HERG region, and which allow specific hybridization to the region. If the target sequence contains a sequence identical to that of the probe, the probes may be short, e.g., in the range of about 8-30 base pairs, since the hybrid will be relatively stable under even stringent conditions. If some degree of mismatch is expected with the probe, i.e., if it is suspected that the probe will hybridize to a variant region, a longer probe may be employed which hybridizes to the target sequence with the requisite specificity.
  • the probes will include an isolated polynucleotide attached to a label or reporter molecule and may be used to isolate other polynucleotide sequences, having sequence similarity by standard methods. For techniques for preparing and labeling probes see, e.g., Sambrook et al., 1989 or Ausubel et al., 1992. Other similar polynucleotides may be selected by using homologous polynucleotides. Alternatively, polynucleotides encoding these or similar polypeptides may be synthesized or selected by use of the redundancy in the genetic code. Various codon substitutions may be introduced, e.g., by silent changes (thereby producing various restriction sites) or to optimize expression for a particular system. Mutations may be introduced to modify the properties of the polypeptide, perhaps to change the polypeptide degradation or turnover rate.
  • Probes comprising synthetic oligonucleotides or other polynucleotides of the present invention may be derived from naturally occurring or recombinant single- or double-stranded polynucleotides, or be chemically synthesized. Probes may also be labeled by nick translation, Klenow fill-in reaction, or other methods known in the art.
  • Portions of the polynucleotide sequence having at least about eight nucleotides, usually at least about 15 nucleotides, and fewer than about 9 kb, usually fewer than about 1.0 kb, from a polynucleotide sequence encoding HERG are preferred as probes.
  • This definition therefore includes probes of sizes 8 nucleotides through 9000 nucleotides.
  • this definition includes probes of 8, 12, 15, 20, 25, 40, 60, 80, 100, 200, 300, 400 or 500 nucleotides or probes having any number of nucleotides within these ranges of values (e.g., 9, 10, 11, 16, 23, 30, 38, 50, 72, 121, etc., nucleotides), or probes having more than 500 nucleotides.
  • the probes may also be used to determine whether mRNA encoding HERG is present in a cell or tissue.
  • the present invention includes all novel probes having at least 8 nucleotides derived from SEQ ID NO:1 or SEQ ID NO:3, its complement or functionally equivalent nucleic acid sequences.
  • the present invention does not include probes which exist in the prior art. That is, the present invention includes all probes having at least 8 nucleotides derived from SEQ ID NO:1 or SEQ ID NO:3 with the proviso that they do not include probes existing in the prior art.
  • primers which may be used for the amplification of all or part of the HERG gene.
  • a definition for primers includes primers of 8, 12, 15, 20, 25, 40, 60, 80, 100, 200, 300, 400, 500 nucleotides, or primers having any number of nucleotides within these ranges of values (e.g., 9, 10, 11, 16, 23, 30, 38, 50, 72, 121, etc. nucleotides), or primers having more than 500 nucleotides, or any number of nucleotides between 500 and 9000.
  • the primers may also be used to determine whether mRNA encoding HERG is present in a cell or tissue.
  • the present invention includes all novel primers having at least 8 nucleotides derived from the HERG locus for amplifying the HERG gene, its complement or functionally equivalent nucleic acid sequences.
  • the present invention does not include primers which exist in the prior art. That is, the present invention includes all primers having at least 8 nucleotides with the proviso that it does not include primers existing in the prior art.
  • Protein modifications or fragments are provided by the present invention for HERG polypeptides or fragments thereof which are substantially homologous to primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate unusual amino acids. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art.
  • a variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as 32 P, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand.
  • radioactive isotopes such as 32 P
  • ligands which bind to labeled antiligands e.g., antibodies
  • fluorophores e.g., chemiluminescent agents
  • enzymes chemiluminescent agents
  • antiligands which can serve as specific binding pair members for a labeled ligand.
  • the choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation.
  • Methods of labeling polypeptides are well known in the art. See Sambrook et al., 1989 or
  • the present invention provides for biologically active fragments of the polypeptides.
  • Significant biological activities include ligand-binding, immunological activity and other biological activities characteristic of HERG polypeptides.
  • Immunological activities include both immunogenic function in a target immune system, as well as sharing of immunological epitopes for binding, serving as either a competitor or substitute antigen for an epitope of the HERG protein.
  • epitope refers to an antigenic determinant of a polypeptide.
  • An epitope could comprise three amino acids in a spatial conformation which is unique to the epitope. Generally, an epitope consists of at least five such amino acids, and more usually consists of at least 8-10 such amino acids. Methods of determining the spatial conformation of such amino acids are known in the art.
  • tandem-repeat polypeptide segments may be used as immunogens, thereby producing highly antigenic proteins.
  • polypeptides will serve as highly efficient competitors for specific binding. Production of antibodies specific for HERG polypeptides or fragments thereof is described below.
  • the present invention also provides for fusion polypeptides, comprising HERG polypeptides and fragments.
  • Homologous polypeptides may be fusions between two or more HERG polypeptide sequences or between the sequences of HERG and a related protein.
  • heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins. For example, ligand-binding or other domains may be “swapped” between different new fusion polypeptides or fragments.
  • Such homologous or heterologous fusion polypeptides may display, for example, altered strength or specificity of binding.
  • Fusion partners include immunoglobulins, bacterial ⁇ -galactosidase, trpE, protein A, ⁇ -lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor. See Godowski et al., 1988.
  • Fusion proteins will typically be made by either recombinant nucleic acid methods, as described below, or may be chemically synthesized. Techniques for the synthesis of polypeptides are described, for example, in Merrifield, 1963.
  • Protein purification refers to various methods for the isolation of the HERG polypeptides from other biological material, such as from cells transformed with recombinant nucleic acids encoding HERG, and are well known in the art.
  • polypeptides may be purified by immunoaffinity chromatography employing, e.g., the antibodies provided by the present invention.
  • immunoaffinity chromatography employing, e.g., the antibodies provided by the present invention.
  • Various methods of protein purification are well known in the art, and include those described in Deutscher, 1990 and Scopes, 1982.
  • isolated is substantially pure when at least about 60 to 75% of a sample exhibits a single polypeptide sequence.
  • a substantially pure protein will typically comprise about 60 to 90% W/W of a protein sample, more usually about 95%, and preferably will be over about 99% pure.
  • Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art which are utilized for purification.
  • a HERG protein is substantially free of naturally associated components when it is separated from the native contaminants which accompany it in its natural state.
  • a polypeptide which is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
  • a protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • a polypeptide produced as an expression product of an isolated and manipulated genetic sequence is an “isolated polypeptide”, as used herein, even if expressed in a homologous cell type. Synthetically made forms or molecules expressed by heterologous cells are inherently isolated molecules.
  • Recombinant nucleic acid is a nucleic acid which is not naturally occurring, or which is made by the artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions.
  • regulatory sequences refers to those sequences normally within 100 kb of the coding region of a locus, but they may also be more distant from the coding region, which affect the expression of the gene (including transcription of the gene, and translation, splicing, stability or the like of the messenger RNA).
  • nucleic acid or fragment thereof is “substantially homologous” (“or substantially similar”) to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases.
  • the percent homology is to be determined using the BLASTN program “BLAST 2 sequences”. This program is available for public use from the National Center for Biotechnology Information (NCBI) over the Internet (http://www.ncbi.nlm.nih.gov/gorf/bl2.html) (Altschul et al., 1997).
  • NCBI National Center for Biotechnology Information
  • the parameters to be used are whatever combination of the following yields the highest calculated percent homology (as calculated below) with the default parameters shown in parentheses:
  • this program shows a percent identity across the complete strands or across regions of the two nucleic acids being matched.
  • the program shows as part of the results an alignment and identity of the two strands being compared. If the strands are of equal length then the identity will be calculated across the complete length of the nucleic acids. If the strands are of unequal lengths, then the length of the shorter nucleic acid is to be used. If the nucleic acids are quite similar across a portion of their sequences but different across the rest of their sequences, the blastn program “BLAST 2 Sequences” will show an identity across only the similar portions, and these portions are reported individually.
  • the percent homology refers to the shorter of the two sequences being compared. If any one region is shown in different alignments with differing percent identities, the alignments which yield the greatest homology are to be used. The averaging is to be performed as in this example of SEQ ID NOs:5 and 6.
  • the program “BLAST 2 Sequences” shows differing alignments of these two nucleic acids depending upon the parameters which are selected. As examples, four sets of parameters were selected for comparing SEQ ID NOs:5 and 6 (gap x_dropoff was 50 for all cases), with the results shown in Table 1. It is to be noted that none of the sets of parameters selected as shown in Table 1 is necessarily the best set of parameters for comparing these sequences. The percent homology is calculated by multiplying for each region showing identity the fraction of bases of the shorter strand within a region times the percent identity for that region and adding all of these together.
  • SEQ ID NO:5 is the short sequence (63 bases), and two regions of identity are shown, the first encompassing bases 4-29 (26 bases) of SEQ ID NO:5 with 92% identity to SEQ ID NO:6 and the second encompassing bases 39-59 (21 bases) of SEQ ID NO:5 with 100% identity to SEQ ID NO:6.
  • Bases 1-3, 30-38 and 60-63 (16 bases) are not shown as having any identity with SEQ ID NO:6.
  • substantial homology or (similarity) exists when a nucleic acid or fragment thereof will hybridize to another nucleic acid (or a complementary strand thereof) under selective hybridization conditions, to a strand, or to its complement.
  • Selectivity of hybridization exists when hybridization which is substantially more selective than total lack of specificity occurs.
  • selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See, Kanehisa, 1984.
  • the length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.
  • Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.
  • Stringent temperature conditions will generally include temperatures in excess of 30° C., typically in excess of 37° C., and preferably in excess of 45° C.
  • Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter.
  • the stringency conditions are dependent on the length of the nucleic acid and the base composition of the nucleic acid and can be determined by techniques well known in the art. See, e.g., Wetmur and Davidson, 1968.
  • substantially homology when referring to polypeptides, indicate that the polypeptide or protein in question exhibits at least about 30% identity with an entire naturally-occurring protein or a portion thereof, usually at least about 70% identity, more usually at least about 80% identity, preferably at least about 90% identity, and more preferably at least about 95% identity.
  • homology for polypeptides, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measures of homology assigned to various substitutions, deletions and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • “Substantially similar function” refers to the function of a modified nucleic acid or a modified protein, with reference to the wild-type HERG nucleic acid or wild-type HERG polypeptide.
  • the modified polypeptide will be substantially homologous to the wild-type HERG polypeptide and will have substantially the same function.
  • the modified polypeptide may have an altered amino acid sequence and/or may contain modified amino acids.
  • the modified polypeptide may have other useful properties, such as a longer half-life.
  • the similarity of function (activity) of the modified polypeptide may be substantially the same as the activity of the wild-type HERG polypeptide.
  • the similarity of function (activity) of the modified polypeptide may be higher than the activity of the wild-type HERG polypeptide.
  • the modified polypeptide is synthesized using conventional techniques, or is encoded by a modified nucleic acid and produced using conventional techniques.
  • the modified nucleic acid is prepared by conventional techniques.
  • a nucleic acid with a function substantially similar to the wild-type HERG gene function produces the modified protein described above.
  • a polypeptide “fragment”, “portion” or “segment” is a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to 13 contiguous amino acids and, most preferably, at least about 20 to 30 or more contiguous amino acids.
  • polypeptides of the present invention may be coupled to a solid-phase support, e.g., nitrocellulose, nylon, column packing materials (e.g., Sepharose beads), magnetic beads, glass wool, plastic, metal, polymer gels, cells, or other substrates.
  • a solid-phase support e.g., nitrocellulose, nylon, column packing materials (e.g., Sepharose beads), magnetic beads, glass wool, plastic, metal, polymer gels, cells, or other substrates.
  • Such supports may take the form, for example, of beads, wells, dipsticks, or membranes.
  • Target region refers to a region of the nucleic acid which is amplified and/or detected.
  • target sequence refers to a sequence with which a probe or primer will form a stable hybrid under desired conditions.
  • polynucleotides of the present invention may be produced by replication in a suitable host cell. Natural or synthetic polynucleotide fragments coding for a desired fragment will be incorporated into recombinant polynucleotide constructs, usually DNA constructs, capable of introduction into and replication in a ptokaryotic or eukaryotic cell. Usually the polynucleotide constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to (with and without integration within the genome) cultured mammalian or plant or other eukaryotic cell lines. The purification of nucleic acids produced by the methods of the present invention are described, e.g., in Sambrook et al., 1989 or Ausubel et al., 1992.
  • the polynucleotides of the present invention may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Caruthers, 1981 or the triester method according to Matteucci and Caruthers, 1981, and may be performed on commercial, automated oligonucleotide synthesizers.
  • a double-stranded fragment may be obtained from the single-stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may comprise a replication system recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment.
  • Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences.
  • ARS origin of replication or autonomously replicating sequence
  • Such vectors may be prepared by means of standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al., 1989 or Ausubel et al., 1992.
  • An appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host, and may include, when appropriate, those naturally associated with HERG genes. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al., 1989 or Ausubel et al., 1992; see also, e.g., Metzger et al., 1988. Many useful vectors are known in the art and may be obtained from such vendors as Stratagene, New England Biolabs, Promega Biotech, and others. Promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts.
  • Useful yeast promoters include promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others. Vectors and promoters suitable for use in yeast expression are further described in Hitzeman et al., EP 73,675A.
  • Non-native mammalian promoters might include the early and late promoters from SV40 (Fiers et al., 1978) or promoters derived from murine Molony leukemia virus, mouse tumor virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma. Insect promoters may be derived from baculovirus.
  • the construct may be joined to an amplifiable gene (e.g., DHFR) so that multiple copies of the gene may be made.
  • DHFR e.g., DHFR
  • Enhancers and Eukaryotic Gene Expression Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1983). See also, e.g., U.S. Pat. Nos. 5,691,198; 5,735,500; 5,747,469 and 5,436,146.
  • expression vectors may replicate autonomously, they may also replicate by being inserted into the genome of the host cell, by methods well known in the art.
  • Expression and cloning vectors will likely contain a selectable marker, a gene encoding a protein necessary for survival or growth of a host cell transformed with the vector. The presence of this gene ensures growth of only those host cells which express the inserts.
  • Typical selection genes encode proteins that a) confer resistance to antibiotics or other toxic substances, e.g. ampicillin, neomycin, methotrexate, etc., b) complement auxotrophic deficiencies, or c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • the choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art.
  • the vectors containing the nucleic acids of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, e.g., by injection (see, Kubo et al., 1988), or the vectors can be introduced directly into host cells by methods well known in the art, which vary depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome); and other methods. See generally, Sambrook et al., 1989 and Ausubel et al., 1992.
  • the introduction of the polynucleotides into the host cell by any method known in the art, including, inter alia, those described above, will be referred to herein as “transformation.”
  • the cells into which have been introduced nucleic acids described above are meant to also include the progeny of such cells.
  • nucleic acids and polypeptides of the present invention may be prepared by expressing HERG nucleic acids or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells.
  • prokaryotic hosts are strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.
  • Mammalian or other eukaryotic host cells such as those of yeast, filamentous fungi, plant, insect, or amphibian or avian species, may also be useful for production of the proteins of the present invention. Propagation of mammalian cells in culture is per se well known. See, Jakoby and Pastan (eds.), 1979. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although it will be appreciated by the skilled practitioner that other cell lines may be appropriate, e.g., to provide higher expression, desirable glycosylation patterns, or other features. An example of a commonly used insect cell line is SF9.
  • Clones are selected by using markers depending on the mode of the vector construction.
  • the marker may be on the same or a different DNA molecule, preferably the same DNA molecule.
  • the transformant may be selected, e.g., by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.
  • Prokaryotic or eukaryotic cells transformed with the polynucleotides of the present invention will be useful not only for the production of the nucleic acids and polypeptides of the present invention, but also, for example, in studying the characteristics of HERG polypeptides.
  • the probes and primers based on the HERG gene sequences disclosed herein are used to identify homologous HERG gene sequences and proteins in other species. These gene sequences and proteins are used in the diagnostic/prognostic, therapeutic and drug screening methods described herein for the species from which they have been isolated.
  • This invention is particularly useful for screening compounds by using the HERG polypeptide or binding fragment thereof in any of a variety of drug screening techniques.
  • the HERG polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, or borne on a cell surface.
  • One method of drug screening utilizes eucaryotic or procaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • One may measure, for example, for the formation of complexes between a HERG polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between a HERG polypeptide or fragment and a known ligand is interfered with by the agent being tested.
  • the present invention provides methods of screening for drugs comprising contacting such an agent with a HERG polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the HERG polypeptide or fragment, or (ii) for the presence of a complex between the HERG polypeptide or fragment and a ligand, by methods well known in the art.
  • the HERG polypeptide or fragment is typically labeled. Free HERG polypeptide or fragment is separated from that present in a protein:protein complex, and the amount of free (i.e., uncomplexed) label is a measure of the binding of the agent being tested to HERG or its interference with HERG:ligand binding, respectively.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the HERG polypeptides and is described in detail in Geysen (published PCT application WO 84/03564). Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with HERG polypeptide and washed. Bound HERG polypeptide is then detected by methods well known in the art.
  • Purified HERG can be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies to the polypeptide can be used to capture antibodies to immobilize the HERG polypeptide on the solid phase.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding the HERG polypeptide compete with a test compound for binding to the HERG polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of the HERG polypeptide.
  • the above screening methods are not limited to assays employing only HERG but are also applicable to studying HERG-protein complexes.
  • the effect of drugs on the activity of this complex is analyzed.
  • the following assays are examples of assays which can be used for screening for drug candidates.
  • a mutant HERG (per se or as part of a fusion protein) is mixed with a wild-type protein (per se or as part of a fusion protein) to which wild-type HERG binds. This mixing is performed in both the presence of a drug and the absence of the drug, and the amount of binding of the mutant HERG with the wild-type protein is measured. If the amount of the binding is more in the presence of said drug than in the absence of said drug, the drug is a drug candidate for treating LQT resulting from a mutation in HERG.
  • a wild-type HERG (per se or as part of a fusion protein) is mixed with a wild-type protein (per se or as part of a fusion protein) to which wild-type HERG binds. This mixing is performed in both the presence of a drug and the absence of the drug, and the amount of binding of the wild-type HERG with the wild-type protein is measured. If the amount of the binding is more in the presence of said drug than in the absence of said drug, the drug is a drug candidate for treating LQT resulting from a mutation in HERG.
  • a mutant protein which as a wild-type protein binds to HERG (per se or as part of a fusion protein) is mixed with a wild-type HERG (per se or as part of a fusion protein). This mixing is performed in both the presence of a drug and the absence of the drug, and the amount of binding of the mutant protein with the wild-type HERG is measured. If the amount of the binding is more in the presence of said drug than in the absence of said drug, the drug is a drug candidate for treating LQT resulting from a mutation in the gene encoding the protein.
  • the polypeptide of the invention may also be used for screening compounds developed as a result of combinatorial library technology.
  • Combinatorial library technology provides an efficient way of testing a potential vast number of different substances for ability to modulate activity of a polypeptide.
  • Such libraries and their use are known in the art.
  • the use of peptide libraries is preferred. See, for example, WO 97/02048.
  • a method of screening for a substance which modulates activity of a polypeptide may include contacting one or more test substances with the polypeptide in a suitable reaction medium, testing the activity of the treated polypeptide and comparing that activity with the activity of the polypeptide in comparable reaction medium untreated with the test substance or substances. A difference in activity between the treated and untreated polypeptides is indicative of a modulating effect of the relevant test substance or substances.
  • test substances Prior to or as well as being screened for modulation of activity, test substances may be screened for ability to interact with the polypeptide, e.g., in a yeast two-hybrid system (e.g., Bartel et al., 1993; Fields and Song, 1989; Chevray and Nathans, 1992; Lee et al., 1995).
  • This system may be used as a coarse screen prior to testing a substance for actual ability to modulate activity of the polypeptide.
  • the screen could be used to screen test substances for binding to an HERG specific binding partner, such as myosin, actinin or dystrophin, or to find mimetics of the HERG polypeptide.
  • the substance may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e., manufacture or formulation, or a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.
  • the present invention extends in various aspects not only to a substance identified using a nucleic acid molecule as a modulator of polypeptide activity, in accordance with what is disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g., for treatment (which may include preventative treatment) of LQT, use of such a substance in the manufacture of a composition for administration, e.g., for treatment of LQT, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
  • a substance identified as a modulator of polypeptide function may be peptide or non-peptide in nature.
  • Non-peptide “small molecules” are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use.
  • the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a “lead” compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g., pure peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
  • Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g., stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g., spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process.
  • a range of sources e.g., spectroscopic techniques, x-ray diffraction data and NMR.
  • Computational analysis, similarity mapping which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms
  • other techniques can be used in this modeling process.
  • the three-dimensional structure of the ligand and its binding partner are modeled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic is peptide-based
  • further stability can be achieved by cyclizing the peptide, increasing its rigidity.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • a biological sample such as blood is prepared and analyzed for the presence or absence of susceptibility alleles of HERG.
  • a biological sample is prepared and analyzed for the presence or absence of mutant alleles of HERG. Results of these tests and interpretive information are returned to the health care provider for communication to the tested individual.
  • diagnoses may be performed by diagnostic laboratories, or, alternatively, diagnostic kits are manufactured and sold to health care providers or to private individuals for self-diagnosis.
  • the screening method involves amplification of the relevant HERG sequences.
  • the screening method involves a non-PCR based strategy.
  • Such screening methods include two-step label amplification methodologies that are well known in the art. Both PCR and non-PCR based screening strategies can detect target sequences with a high level of sensitivity.
  • the most popular method used today is target amplification.
  • the target nucleic acid sequence is amplified with polymerases.
  • One particularly preferred method using polymerase-driven amplification is the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the polymerase chain reaction and other polymerase-driven amplification assays can achieve over a million-fold increase in copy number through the use of polymerase-driven amplification cycles.
  • the resulting nucleic acid can be sequenced or used as a substrate for DNA probes.
  • the biological sample to be analyzed such as blood or serum
  • the sample nucleic acid may be prepared in various ways to facilitate detection of the target sequence, e.g. denaturation, restriction digestion, electrophoresis or dot blotting.
  • the targeted region of the analyte nucleic acid usually must be at least partially single-stranded to form hybrids with the targeting sequence of the probe. If the sequence is naturally single-stranded, denaturation will not be required. However, if the sequence is double-stranded, the sequence will probably need to be denatured. Denaturation can be carried out by various techniques known in the art.
  • Analyte nucleic acid and probe are incubated under conditions which promote stable hybrid formation of the target sequence in the probe with the putative targeted sequence in the analyte.
  • the region of the probes which is used to bind to the analyte can be made completely complementary to the targeted region of human chromosome 7. Therefore, high stringency conditions are desirable in order to prevent false positives. However, conditions of high stringency are used only if the probes are complementary to regions of the chromosome which are unique in the genome.
  • the stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, base composition, probe length, and concentration of formamide.
  • Detection, if any, of the resulting hybrid is usually accomplished by the use of labeled probes.
  • the probe may be unlabeled, but may be detectable by specific binding with a ligand which is labeled, either directly or indirectly.
  • Suitable labels, and methods for labeling probes and ligands are known in the art, and include, for example, radioactive labels which may be incorporated by known methods (e.g., nick translation, random priming or kinasing), biotin, fluorescent groups, chemiluminescent groups (e.g., dioxetanes, particularly triggered dioxetanes), enzymes, antibodies, gold nanoparticles and the like.
  • Variations of this basic scheme are known in the art, and include those variations that facilitate separation of the hybrids to be detected from extraneous materials and/or that amplify the signal from the labeled moiety. A number of these variations are reviewed in, e.g., Matthews and Kricka, 1988; Landegren et al., 1988; Mifflin, 1989; U.S. Pat. No. 4,868,105; and in EPO Publication No. 225,807.
  • non-PCR based screening assays are also contemplated in this invention.
  • This procedure hybridizes a nucleic acid probe (or an analog such as a methyl phosphonate backbone replacing the normal phosphodiester), to the low level DNA target.
  • This probe may have an enzyme covalently linked to the probe, such that the covalent linkage does not interfere with the specificity of the hybridization.
  • This enzyme-probe-conjugate-target nucleic acid complex can then be isolated away from the free probe enzyme conjugate and a substrate is added for enzyme detection. Enzymatic activity is observed as a change in color development or luminescent output resulting in a 10 3 -10 6 increase in sensitivity.
  • Jablonski et al., 1986 see Jablonski et al., 1986.
  • Two-step label amplification methodologies are known in the art. These assays work on the principle that a small ligand (such as digoxigenin, biotin, or the like) is attached to a nucleic acid probe capable of specifically binding HERG. Allele specific probes are also contemplated within the scope of this example and exemplary allele specific probes include probes encompassing the predisposing mutations of this disclosure.
  • a small ligand such as digoxigenin, biotin, or the like
  • Allele specific probes are also contemplated within the scope of this example and exemplary allele specific probes include probes encompassing the predisposing mutations of this disclosure.
  • the small ligand attached to the nucleic acid probe is specifically recognized by an antibody-enzyme conjugate.
  • digoxigenin is attached to the nucleic acid probe. Hybridization is detected by an antibody-alkaline phosphatase conjugate which turns over a chemiluminescent substrate.
  • the small ligand is recognized by a second ligand-enzyme conjugate that is capable of specifically complexing to the first ligand.
  • a well known embodiment of this example is the biotin-avidin type of interactions. For methods for labeling nucleic acid probes and their use in biotin-avidin based assays see Rigby et al., 1977 and Nguyen et al., 1992.
  • the nucleic acid probe assays of this invention will employ a cocktail of nucleic acid probes capable of detecting HERG.
  • a cocktail of nucleic acid probes capable of detecting HERG in one example to detect the presence of HERG in a cell sample, more than one probe complementary to the gene is employed and in particular the number of different probes is alternatively two, three, or five different nucleic acid probe sequences.
  • the cocktail includes probes capable of binding to the allele-specific mutations identified in populations of patients with alterations in HERG.
  • any number of probes can be used, and will preferably include probes corresponding to the major gene mutations identified as predisposing an individual to LQT.
  • LQT can also be detected on the basis of the alteration of wild-type HERG polypeptide. Such alterations can be determined by sequence analysis in accordance with conventional techniques. More preferably, antibodies (polyclonal or monoclonal) are used to detect differences in, or the absence of HERG peptides. Techniques for raising and purifying antibodies are well known in the art and any such techniques may be chosen to achieve the preparations claimed in this invention. In a preferred embodiment of the invention, antibodies will immunoprecipitate HERG proteins from solution as well as react with these proteins on Western or immunoblots of polyacrylamide gels. In another preferred embodiment, antibodies will detect HERG proteins in paraffin or frozen tissue sections, using immunocytochemical techniques.
  • Preferred embodiments relating to methods for detecting HERG or their mutations include enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies.
  • ELISA enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • IRMA immunoradiometric assays
  • IEMA immunoenzymatic assays
  • sandwich assays are described by David et al., in U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby incorporated by reference.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson, 1991.
  • peptides e.g., HERG polypeptide
  • an amino acid residue is replaced by Ala, and its effect on the peptide's activity is determined.
  • Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
  • drugs which have, e.g., improved HERG polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of HERG polypeptide activity.
  • sufficient amounts of the HERG polypeptide may be made available to perform such analytical studies as x-ray crystallography.
  • the knowledge of the HERG protein sequences provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.
  • a method is also provided of supplying wild-type HERG function to a cell which carries mutant HERG alleles. Supplying such a function should allow normal functioning of the recipient cells.
  • the wild-type gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. More preferred is the situation where the wild-type gene or a part thereof is introduced into the mutant cell in such a way that it recombines with the endogenous mutant gene present in the cell. Such recombination requires a double recombination event which results in the correction of the gene mutation.
  • Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector may be used.
  • Methods for introducing DNA into cells such as electroporation, calcium phosphate co-precipitation and viral transduction are known in the art, and the choice of method is within the competence of the practitioner.
  • the HERG gene or fragment may be employed in gene therapy methods in order to increase the amount of the expression products of such genes in cells. It may also be useful to increase the level of expression of a given LQT gene even in those heart cells in which the mutant gene is expressed at a “normal” level, but the gene product is not fully functional.
  • Gene therapy would be carried out according to generally accepted methods, for example, as described by Friedman (1991) or Culver (1996).
  • Cells from a patient would be first analyzed by the diagnostic methods described above, to ascertain the production of HERG polypeptide in the cells.
  • a virus or plasmid vector (see further details below), containing a copy of the HERG gene linked to expression control elements and capable of replicating inside the cells, is prepared.
  • the vector may be capable of replicating inside the cells.
  • the vector may be replication deficient and is replicated in helper cells for use in gene therapy.
  • Suitable vectors are known, such as disclosed in U.S. Pat. No. 5,252,479 and published PCT application WO 93107282 and U.S. Pat. Nos. 5,691,198; 5,747,469; 5,436,146 and 5,753,500.
  • the vector is then injected into the patient. If the transfected gene is not permanently incorporated into the genome of each of the targeted cells, the treatment may have to
  • Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and nonviral transfer methods.
  • viruses have been used as gene transfer vectors or as the basis for preparing gene transfer vectors, including papovaviruses (e.g., SV40, Madzak et al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson and Akrigg, 1992; Stratford-Perricaudet et al., 1990; Schneider et al., 1998), vaccinia virus (Moss, 1992; Moss, 1996), adeno-associated virus (Muzyczka, 1992; Ohi et al., 1990; Russell and Hirata, 1998), herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et al
  • Nonviral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham and van der Eb, 1973; Pellicer et al., 1980); mechanical techniques, for example microinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster et al., 1981; Costantini and Lacy, 1981); membrane fusion-mediated transfer via liposomes (Felgner et al., 1987; Wang and Huang, 1989; Kaneda et al., 1989; Stewart et al., 1992; Nabel et al., 1990; Lim et al., 1991); and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al., 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al., 1989; Wolff et al., 1991; Wagner et al., 1990; Wagner et al., 1991; Cotten et al., 1990
  • Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery, allowing one to direct the viral vectors to the tumor cells and not into the surrounding nondividing cells.
  • the retroviral vector producer cell line can be injected into tumors (Culver et al., 1992). Injection of producer cells would then provide a continuous source of vector particles. This technique has been approved for use in humans with inoperable brain tumors.
  • plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein, and the resulting complex is bound to an adenovirus vector.
  • the trimolecular complex is then used to infect cells.
  • the adenovirus vector permits efficient binding, internalization, and degradation of the endosome before the coupled DNA is damaged.
  • adenovirus based vectors see Schneider et al. (1998) and U.S. Pat. Nos. 5,691,198; 5,747,469; 5,436,146 and 5,753,500.
  • Liposome/DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is nonspecific, localized in vivo uptake and expression have been reported in tumor deposits, for example, following direct in situ administration (Nabel, 1992).
  • Expression vectors in the context of gene therapy are meant to include those constructs containing sequences sufficient to express a polynucleotide that has been cloned therein.
  • the construct contains viral sequences sufficient to support packaging of the construct. If the polynucleotide encodes HERG, expression will produce HERG. If the polynucleotide encodes an antisense polynucleotide or a ribozyme, expression will produce the antisense polynucleotide or ribozyme. Thus in this context, expression does not require that a protein product be synthesized.
  • the vector also contains a promoter functional in eukaryotic cells.
  • the cloned polynucleotide sequence is under control of this promoter. Suitable eukaryotic promoters include those described above.
  • the expression vector may also include sequences, such as selectable markers and other sequences described herein.
  • Receptor-mediated gene transfer is accomplished by the conjugation of DNA (usually in the form of covalently closed supercoiled plasmid) to a protein ligand via polylysine.
  • Ligands are chosen on the basis of the presence of the corresponding ligand receptors on the cell surface of the target cell/tissue type.
  • These ligand-DNA conjugates can be injected directly into the blood if desired and are directed to the target tissue where receptor binding and internalization of the DNA-protein complex occurs.
  • coinfection with adenovirus can be included to disrupt endosome function.
  • the therapy is as follows: patients who carry a HERG susceptibility allele are treated with a gene delivery vehicle such that some or all of their heart precursor cells receive at least one additional copy of a functional normal HERG allele. In this step, the treated individuals have reduced risk of LQT to the extent that the effect of the susceptible allele has been countered by the presence of the normal allele.
  • Peptides which have HERG activity can be supplied to cells which carry a mutant or missing HERG allele.
  • Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors.
  • HERG polypeptide can be extracted from HERG-producing mammalian cells.
  • the techniques of synthetic chemistry can be employed to synthesize HERG protein. Any of such techniques can provide the preparation of the present invention which comprises the HERG protein. The preparation is substantially free of other human proteins. This is most readily accomplished by synthesis in a microorganism or in vitro.
  • Active HERG molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some active molecules may be taken up by cells, actively or by diffusion. Supply of molecules with HERG activity should lead to partial reversal of LQT. Other molecules with HERG activity (for example, peptides, drugs or organic compounds) may also be used to effect such a reversal. Modified polypeptides having substantially similar function are also used for peptide therapy.
  • Animals for testing therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. Such treatments include insertion of mutant HERG alleles, usually from a second animal species, as well as insertion of disrupted homologous genes.
  • the endogenous HERG gene(s) of the animals may be disrupted by insertion or deletion mutation or other genetic alterations using conventional techniques (Capecchi, 1989; Valancius and Smithies, 1991; Hasty et al., 1991; Shinkai et al., 1992; Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992; Donehower et al., 1992).
  • test substances After test substances have been administered to the animals, the presence of LQT must be assessed. If the test substance prevents or suppresses the appearance of LQT, then the test substance is a candidate therapeutic agent for treatment of LQT.
  • test substances After test substances have been administered to the animals, the presence of LQT must be assessed. If the test substance prevents or suppresses the appearance of LQT, then the test substance is a candidate therapeutic agent for treatment of LQT.
  • HERG alleles are screened for mutations either directly or after cloning the alleles.
  • the alleles are tested for the presence of nucleic acid sequence differences from the normal allele using any suitable technique, including but not limited to, one of the following methods: fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single stranded conformation analysis (SSCP), linkage analysis, RNase protection assay, allele specific oligonucleotide (ASO), dot blot analysis and PCR-SSCP analysis.
  • FISH fluorescent in situ hybridization
  • PFGE analysis direct DNA sequencing
  • SSCP single stranded conformation analysis
  • ASO allele specific oligonucleotide
  • RNA transcripts of the HERG gene or gene fragment are hybridized to single stranded whole genomic DNA from an individual to be tested, and the resulting heteroduplex is treated with Ribonuclease A (RNase A) and run on a denaturing gel to detect the location of any mismatches.
  • RNase A Ribonuclease A
  • the alleles of the HERG gene in an individual to be tested are cloned using conventional techniques. For example, a blood sample is obtained from the individual. The genomic DNA isolated from the cells in this sample is partially digested to an average fragment size of approximately 20 kb. Fragments in the range from 18-21 kb are isolated. The resulting fragments are ligated into an appropriate vector. The sequences of the clones are then determined and compared to the normal HERG gene.
  • PCRs polymerase chain reactions
  • primer pairs for the 5′ region or the exons of the HERG gene are performed with primer pairs for the 5′ region or the exons of the HERG gene.
  • PCRs can also be performed with primer pairs based on any sequence of the normal HERG gene.
  • primer pairs for one of the introns can be prepared and utilized.
  • RT-PCR can also be performed on the mRNA.
  • the amplified products are then analyzed by single stranded conformation polymorphisms (SSCP) using conventional techniques to identify any differences and these are then sequenced and compared to the normal gene sequence.
  • SSCP single stranded conformation polymorphisms
  • Individuals can be quickly screened for common HERG gene variants by amplifying the individual's DNA using suitable primer pairs and analyzing the amplified product, e.g., by dot-blot hybridization using allele-specific oligonucleotide probes.
  • the second method employs RNase A to assist in the detection of differences between the normal HERG gene and defective genes. This comparison is performed in steps using small ( ⁇ 500 bp) restriction fragments of the HERG gene as the probe.
  • the HERG gene is digested with a restriction enzyme(s) that cuts the gene sequence into fragments of approximately 500 bp. These fragments are separated on an electrophoresis gel, purified from the gel and cloned individually, in both orientations, into an SP6 vector (e.g., pSP64 or pSP65).
  • the SP6-based plasmids containing inserts of the HERG gene fragments are transcribed in vitro using the SP6 transcription system, well known in the art, in the presence of [ ⁇ - 32 P]GTP, generating radiolabeled RNA transcripts of both strands of the gene.
  • RNA transcripts are used to form heteroduplexes with the allelic DNA using conventional techniques.
  • Mismatches that occur in the RNA:DNA heteroduplex owing to sequence differences between the HERG fragment and the HERG allele subclone from the individual, result in cleavage in the RNA strand when treated with RNase A.
  • Such mismatches can be the result of point mutations or small deletions in the individual's allele. Cleavage of the RNA strand yields two or more small RNA fragments, which run faster on the denaturing gel than the RNA probe itself.
  • any differences which are found, will identify an individual as having a molecular variant of the HERG gene and the consequent presence of LQT.
  • These variants can take a number of forms. The most severe forms would be frame shift mutations or large deletions which would cause the gene to code for an abnormal protein or one which would significantly alter protein expression. Less severe disruptive mutations would include small in-flame deletions and nonconservative base pair substitutions which would have a significant effect on the protein produced, such as changes to or from a cysteine residue, from a basic to an acidic amino acid or vice versa, from a hydrophobic to hydrophilic amino acid or vice versa, or other mutations which would affect secondary or tertiary protein structure. Silent mutations or those resulting in conservative amino acid substitutions would not generally be expected to disrupt protein function.
  • the HERG polypeptides, antibodies, peptides and nucleic acids of the present invention can be formulated in pharmaceutical compositions, which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.).
  • the composition may contain the active agent or pharmaceutically acceptable salts of the active agent.
  • These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, intrathecal, epineural or parenteral.
  • the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
  • the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, WO 96/11698.
  • the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
  • suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
  • the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
  • the compounds When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
  • the active agent is preferably administered in a therapeutically effective amount.
  • the actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington 's Pharmaceutical Sciences.
  • targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
  • these agents could be produced in the target cell, e.g. in a viral vector such as described above or in a cell based delivery system such as described in U.S. Pat. No. 5,550,050 and published PCT application Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635, designed for implantation in a patient.
  • the vector could be targeted to the specific cells to be treated, or it could contain regulatory elements which are more tissue specific to the target cells.
  • the cell based delivery system is designed to be implanted in a patient's body at the desired target site and contains a coding sequence for the active agent.
  • the agent could be administered in a precursor form for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. See for example, EP 425,731A and WO 90/07936.
  • LQT LQT
  • mutations in specific genes e.g. HERG
  • treatments with any of a variety of drugs can also cause LQT.
  • These drugs include those being taken to treat cardiac arrhythmias and also other drugs including antihistamines and some antibiotics such as erythromycin.
  • LQT is a result of mutations (hereditary or familial LQT) or drug induced (acquired LQT)
  • the drugs interact with the K + channel I Kr , the major subunit of which is encoded by HERG, thereby affecting K + flow in cardiac cells. Mutations in HERG also can affect K + flow through this channel.
  • SCN5A contains four homologous domains (DI-DIV), each of which contains six putative membrane spanning segments (S1-S6).
  • DI-DIV homologous domains
  • S1-S6 putative membrane spanning segments
  • HERG a cardiac potassium channel gene
  • hereditary LQT The mutations identified in HERG, and the biophysics of potassium channel alpha subunits, suggest that chromosome 7-linked hereditary LQT results from dominant-negative mutations and a resultant reduction in functional channels.
  • LQT Presymptomatic diagnosis of LQT has depended on identification of QT prolongation on electrocardiograms. Unfortunately, electrocardiograms are rarely performed in young, healthy individuals. In addition, many LQT gene carriers have relatively normal QT intervals, and the first sign of disease can be a fatal cardiac arrhythmia (Vincent et al., 1992). Now that four LQT genes have been identified, genetic testing for this disorder can be contemplated. This will require continued mutational analyses and identification of additional LQT genes. With more detailed phenotypic analyses, phenotypic differences between the varied forms of LQT may be discovered. These differences may be useful for diagnosis and treatment.
  • the identification of the association between the HERG, KVLQT1, SCN5A and KCNE1 gene mutations and hereditary LQT permits the early presymptomatic screening of individuals to identify those at risk for developing LQT. To identify such individuals, the alleles are screened for mutations either directly or after cloning the alleles.
  • the alleles are tested for the presence of nucleic acid sequence differences from the normal allele using any suitable technique, including but not limited to, one of the following methods: fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single stranded conformation analysis (SSCP), linkage analysis, RNase protection assay, allele specific oligonucleotide (ASO) dot blot analysis and PCR-SSCP analysis.
  • FISH fluorescent in situ hybridization
  • direct DNA sequencing PFGE analysis
  • Southern blot analysis Southern blot analysis
  • SSCP single stranded conformation analysis
  • linkage analysis RNase protection assay
  • ASO allele specific oligonucleotide
  • RNA transcripts of the HERG gene or gene fragment are hybridized to single stranded whole genomic DNA from an individual to be tested, and the resulting heteroduplex is treated with Ribonuclease A (RNase A) and run on a denaturing gel to detect the location of any mismatches.
  • RNase A Ribonuclease A
  • the alleles of the HERG gene in an individual to be tested are cloned using conventional techniques. For example, a blood sample is obtained from the individual. The genomic DNA isolated from the cells in this sample is partially digested to an average fragment size of approximately 20 kb. Fragments in the range from 18-21 kb are isolated. The resulting fragments are ligated into an appropriate vector. The sequences of the clones are then determined and compared to the normal HERG gene.
  • PCRs polymerase chain reactions
  • primer pairs for the 5′ region or the exons of the HERG gene are performed with primer pairs for the 5′ region or the exons of the HERG gene.
  • PCRs can also be performed with primer pairs based on any sequence of the normal HERG gene.
  • primer pairs for one of the introns can be prepared and utilized.
  • PCR can also be performed on the mRNA.
  • the amplified products are then analyzed by single stranded conformation polymorphisms (SSCP) using conventional techniques to identify any differences and these are then sequenced and compared to the normal gene sequence.
  • SSCP single stranded conformation polymorphisms
  • Individuals can be quickly screened for common HERG gene variants by amplifying the individual's DNA using suitable primer pairs and analyzing the amplified product, e.g., by dot-blot hybridization using allele-specific oligonucleotide probes.
  • the second method employs RNase A to assist in the detection of differences between the normal HERG gene and defective genes. This comparison is performed in steps using small ( ⁇ 500 bp) restriction fragments of the HERG gene as the probe.
  • the HERG gene is digested with a restriction enzyme(s) that cuts the gene sequence into fragments of approximately 500 bp. These fragments are separated on an electrophoresis gel, purified from the gel and cloned individually, in both orientations, into an SP6 vector (e.g., pSP64 or pSP65).
  • the SP6-based plasmids containing inserts of the HERG gene fragments are transcribed in vitro using the SP6 transcription system, well known in the art, in the presence of [ ⁇ - 32 P]GTP, generating radiolabeled RNA transcripts of both strands of the gene.
  • RNA transcripts are used to form heteroduplexes with the allelic DNA using conventional techniques.
  • Mismatches that occur in the RNA:DNA heteroduplex owing to sequence differences between the HERG fragment and the HERG allele subclone from the individual, result in cleavage in the RNA strand when treated with RNase A.
  • Such mismatches can be the result of point mutations or small deletions in the individual's allele. Cleavage of the RNA strand yields two or more small RNA fragments, which run faster on the denaturing gel than the RNA probe itself.
  • any differences which are found, will identify an individual as having a molecular variant of the HERG gene and the consequent presence of long QT syndrome. These variants can take a number of forms. The most severe forms would be frame shift mutations or large deletions which would cause the gene to code for an abnormal protein or one which would significantly alter protein expression. Less severe disruptive mutations would include small in-frame deletions and nonconservative base pair substitutions which would have a significant effect on the protein produced, such as changes to or from a cysteine residue, from a basic to an acidic amino acid or vice versa, from a hydrophobic to hydrophilic amino acid or vice versa, or other mutations which would affect secondary or tertiary protein structure. Silent mutations or those resulting in conservative amino acid substitutions would not generally be expected to disrupt protein function.
  • HERG Encodes a K + Channel with Inward Rectification Properties Similar to I Kr .
  • HERG current was activated in response to test potentials > ⁇ 50 mV.
  • the magnitude of HERG current increased progressively with test potentials up to ⁇ 10 mV (FIG. 1 A), then progressively decreased with test potentials ⁇ 0 mV (FIG. 1 B).
  • Deactivation of current tail current was assessed after return of the membrane to the holding potential of ⁇ 70 mV.
  • the amplitude of the tail currents progressively increased after depolarization and saturated at +10 mV.
  • the HERG current-voltage (I-V) relationship determined for 10 oocytes is shown in FIG. 1 C. Peak outward current decreased with incremental depolarization, indicating that HERG is an inward rectifier.
  • the voltage-dependence of channel activation was assessed by plotting the relative amplitude of tail currents as a function of test potential (FIG. 1 D). HERG reached half-maximal activation at a potential of ⁇ 1 5.1 mV.
  • HERG as a delayed rectifier K + channel with a voltage-dependence of activation and rectification properties nearly identical to I Kr (Sanguinetti and Jurkiewicz, 1990; Shibasaki, 1987; Yang et al., 1994). These properties are unlike any other cardiac current.
  • HERG time-course of current activation and deactivation was determined.
  • the time-course for the onset of current (activation) was best fit with a single exponential function (FIG. 2 A).
  • the rate of activation increased with incremental changes in test potentials from ⁇ 40 to +50 mV.
  • Deactivating currents were best fit with a biexponential function (FIG. 2 B), similar to I Kr (Chinn, 1993; Yang et al., 1994).
  • the relative amplitude of the fast component of deactivation varied from 0.77 at ⁇ 30 mV to 0.2 at ⁇ 120 mV (FIG. 2 D).
  • the kinetics of HERG current are slower than I Kr (Sanguinetti and Jurkiewicz, 1990; Shibasaki, 1987; Yang et al., 1994), but exhibit an identical voltage-dependence.
  • HERG Current is Activated by Extracellular K + .
  • the K + -selectivity of HERG was determined by measuring the reversal potential of currents in oocytes bathed in ND96 solution containing different concentrations of KCl (0.5-20 mM). Tail currents were measured at a variable test potential after current activation by a pulse to +20 mV (FIGS. 3 A and 3 B). The voltage at which the tail current reversed from an inward to an outward current was defined as the reversal potential, E rev . This varied with extracellular K + concentration ([K + ] e ), as predicted by the Nernst equation (58 mV change for a 10-fold increase in [K + ]e) for [K + ] e ⁇ 5 mM.
  • FIGS. 4A-C A hallmark feature of cardiac I Kr is its modulation by [K + ] e (Sanguinetti and Jurkiewicz, 1992).
  • the effect of [K + ] e on the magnitude of HERG current is shown in FIGS. 4A-C.
  • HERG current increased in direct proportion to [K + ] e , although the shape of the I-V relationship was not altered (FIG. 4 D).
  • the [K + ] e -dependence of HERG current was determined by comparing the peak outward current at +20 mV in oocytes bathed in solutions containing 0.5 to 20 mM KCl. Over this range, HERG current amplitude varied as a linear function of [K +] e (FIG. 4 E). Unlike most other K + currents, the magnitude of outward HERG current is paradoxically reduced upon removal of extracellular K + .
  • Tail currents were recorded at several test potentials, each preceded by a prepulse to +40 mV (FIG. 5 A).
  • the voltage-dependence of the time constant for recovery from fast inactivation is plotted in FIG. 5 B.
  • the bell-shaped relationship between the time constant for recovery from inactivation and membrane potential peaked at the same voltage ( ⁇ 30 mV) as the relationship describing the voltage-dependence of HERG current activation and deactivation (FIG. 2 C).
  • the onset of fast inactivation could not be quantified (because it occurred much faster than activation), it is likely that the descending limb of the curve in FIG. 4B (from ⁇ 20 to +20 mV) also describes the voltage-dependence of rapid inactivation.
  • the voltage-dependence of channel rectification was determined by comparison of the fully-activated I-V relationship for HERG current (FIG. 5C) with the I-V relationship expected for an ohmic conductor.
  • the dotted line in FIG. 5C was extrapolated from a linear fit of current amplitudes measured at ⁇ 90 to ⁇ 120 mV, and described the I-V relationship that would occur in the absence of inward rectification (ohmic conduction).
  • the slope of this line defined the maximal conductance of HERG in this oocyte (118 ⁇ S), and was used to calculate the voltage-dependence of channel rectification (FIG. 5 D).
  • Rectification was half-maximal at ⁇ 49 mV, and the relationship had a slope factor of 28 mV.
  • the half-point was very similar to I Kr in rabbit nodal cells and the slope factor was nearly identical to I Kr in guinea pig myocytes (Table 2).
  • V t Steady-state HERG current at any given test potential
  • I HERG G ⁇ n ⁇ R ⁇ ( V t ⁇ E rev )
  • G maximal conductance of HERG current
  • n activation variable
  • R rectification variable
  • E rev reversal potential
  • the HERG channel contains a segment homologous to a cyclic nucleotide binding domain near its carboxyl terminus (Wannke and Ganetzky, 1994).
  • 8-Br-cAMP and 8-Br-cGMP were tested.
  • These membrane permeant analogs of endogenous cyclic nucleotides have been shown to increase the magnitude of other channels expressed in Xenopus oocytes (Blumenthal and Kaczmarek, 1992; Bruggemann et al., 1993). Neither compound had a significant effect on current magnitude or voltage-dependence of channel activation at a concentration of 1 mM within 30 min of application (data not shown).
  • the above results show that HERG encodes the major subunit of the cardiac I Kr channel.
  • HERG expressed in oocytes induces a current that shares most of the distinguishing characteristics defining I Kr in cardiac myocytes (Table 2). These include: 1) inward rectification of the I-V relationship, with a peak near 0 mV; 2) voltage dependence of activation; 3) paradoxical modulation of current by [K + ] e ; and 4) block by La 3+ and Co 2+ .
  • the kinetics of activation and deactivation of HERG current are much slower than I Kr in mouse AT-1 cells measured at room temperature (Yang, et al., 1994).
  • HERG is not activated by 8-Br-cAMP, consistent with the finding that isoproterenol does not increase I Kr in cardiac myocytes (Sanguinetti et al., 1991). Co-assembly of HERG subunits in oocytes, presumedly as homotetramers (MacKinnon, 1991), therefore, can reconstitute the major biophysical properties of cardiac I Kr . No other channel shares all these characteristics.
  • HERG is not blocked by methanesulfonanilide drugs (E-4031, MK-499), potent and specific blockers of I Kr in isolated cardiac myocytes (Lynch et al., 1994; Sanguinetti and Jurkiewicz, 1990). This suggests that the I Kr channel and the methanesulfonanilide receptor are separate, but interacting, proteins.
  • K ATP methanesulfonanilide drugs
  • this channel When this channel (rcK ATP -1) is expressed in HEK293 cells, it has all the biophysical characteristics of the native channel (Ashford et al., 1994), including modulation by intracellular nucleotides. However, the channel is not blocked by glibenclamide, a drug that inhibits K ATP channels in cardiac myocytes (Ashford et al., 1994). It may be possible to isolate the methanesulfonanilide receptor biochemically using known high affinity probes such as dofetilide or MK-499. Co-expression of HERG channels with the methanesulfonanilide receptor will enable detailed studies of the interaction between these two molecules.
  • I Kr inward rectification of the I-V relationship.
  • the cardiac inward rectifier, I K1 also exhibits intense inward rectification, but this occurs over a much more negative voltage range.
  • peak outward I K1 occurs at ⁇ 60 mV, whereas I Kr peaks at 0 mV.
  • the mechanism of I K1 rectification results from both a voltage-dependent gating mechanism and block of outward current by intracellular Mg 2+ (Vandenberg, 1987) and spermine (Fakler et al., 1995).
  • inward rectification of I Kr results from voltage-dependent inactivation that occurs much faster than activation (Shibasaki, 1987).
  • Rectification of HERG current was half-maximal (V 1 ⁇ 2 ) at ⁇ 49 mV, and had a slope factor of 28 mV.
  • the slope factor of HERG rectification was similar to I Kr measured in guinea pig myocytes (22 mV).
  • the V 1 ⁇ 2 of HERG rectification was more negative than that estimated in guinea pig (Table 2).
  • the voltage-dependence of I Kr rectification in guinea pig myocytes was difficult to measure because of overlap with a much larger I K1 at negative test potentials.
  • HERG (and I Kr ) by [K + ] e may have physiologic importance.
  • K + accumulates within intracellular clefts (Gintant et al., 1992). This elevation in [K + ] e would increase the contribution of HERG (I Kr ) to net repolarizing current.
  • HERG (I Kr ) may be even more important, therefore, in modulation of action potential duration at high heart rates, or during the initial phase of ischemia.
  • Inherited LQT and the more common (drug-induced) acquired form of the disorder, are associated with torsade de pointes, a polymorphic ventricular tachyarrhythmia. It was recently shown that mutations in HERG cause chromosome 7-linked LQT, likely by a dominant-negative inhibition of HERG function (Curran et al., 1995). It should be noted that there are likely to be several different mechanisms that account for acquired and inherited LQT. For example, it was recently demonstrated that mutations in SCN5A, the cardiac sodium channel gene, cause chromosome 3-linked LQT (Wang et al., 1995). The discovery that HERG forms the I Kr channel provides a logical explanation for the observation that block of I Kr by certain drugs can provoke the same arrhythmia (torsade de pointes) as observed in familial LQT.
  • Medications e.g., sotalol, dofetilide
  • I Kr can be effective antiarrhythmic agents because they modestly lengthen cardiac action potentials, thereby inhibiting re-entrant arrhythmias.
  • hypokalemia In the setting of hypokalemia, however, this effect would be exaggerated, leading to excessive action potential prolongation and induction of torsade de pointes.
  • LQT e.g., antihistamines or antibiotics such as erythromycin
  • HERG encodes the major subunit forming I Kr channels. This discovery suggests that the molecular mechanism of chromosome 7-linked LQT, and certain acquired forms of the disorder, can result from dysfunction of the same ion channel.
  • LQT kindreds were ascertained from medical clinics throughout North America. Phenotypic criteria were identical to those used in previous studies (Keating et al., 1991a; Keating et al., 1991b; Keating, 1992). Individuals were evaluated for LQT based on the QT interval corrected for heart rate (QTc; Bazette, 1920), and the presence of syncope, seizures, and aborted sudden death. Informed consent was obtained from each individual, or their guardians, in accordance with local institutional review board guidelines. Phenotypic data were interpreted without knowledge of genotype. Symptomatic individuals with a corrected QT interval (QTc) of 0.45 seconds or greater and asymptomatic individuals with a QTc of 0.47 seconds or greater were classified as affected. Asymptomatic individuals with a QTc of 0.41 seconds or less were classified as unaffected. Asymptomatic individuals with QTc between 0.41 and 0.47 seconds and symptomatic individuals with QTc of 0.44 seconds or less were classified as uncertain.
  • Pairwise linkage analysis was performed using MLINK in LINKAGE v5.1 (Lathrop et al., 1985). Assumed values of 0.90 for penetrance and 0.001 for LQT gene frequency were used. Gene frequency was assumed to be equal between males and females.
  • HERG probes were generated using the products of PCR reactions with human genomic DNA and primer pairs 1-10, 6-13 and 15-17 (Table 3). These products were cloned, radiolabeled to high specific activity and used to screen a human genomic P1 library (Sternberg, 1990). Positive clones were purified, characterized and used for FISH and DNA sequence analyses. A HERG genomic clone containing domains S1-S3 and intron I (Curran et al., 1995) (intron 6 here) was used to screen ⁇ 10 6 recombinants of a human hippocampal cDNA library (Stratagene, library #936205).
  • a single, partially processed cDNA clone that contained nucleotides 32-2398 of HERG coding sequence was identified.
  • a second screen of this library was performed using the coding portion of this cDNA. This screen produced a second clone containing HERG coding sequence from nucleotides 1216 through the 3′ untranslated region (UTR), and included a poly-A+ region. These two cDNAs were ligated using an XhoI site at position 2089.
  • ⁇ 10 6 clones of a human heart cDNA library (Stratagene, library #936207) were screened with the composite hippocampal cDNA.
  • a single clone containing the 5′-UTR through nucleotide 2133 was isolated. This clone was combined with the hippocampal composite at a BglII site (nucleotide 1913) to produce a full-length HERG cDNA.
  • a PCR assay specific for the 3′ untranslated region of HERG (employing primers 5′GCTGGGCCGCTCCCCTTGGA3′ (SEQ ID NO:7) and 5′GCATCTTCATTAATTATTCA3′ (SEQ ID NO:8) and yielding a 309-bp product) was used to screen a collection of YAC clones highly enriched for human chromosome 7 (Green et al., in press). Two positive YAC clones were identified (yWSS2193 and yWSS1759), both were contained within a larger contig that includes YACs positive for the genetic marker D7S505 (Green et al., 1994).
  • Metaphase chromosome spreads were prepared from normal cultured lymphocytes (46 X,Y) by standard procedures of colcemid arrest, hypotonic treatment and acetic acid-methanol fixation.
  • HERG P1 clone 16B4 was labeled by incorporation of biotin-14-dATP (BioNick System, Gibco-BRL), hybridized to metaphase spreads and detected with streptavidin-Cy3 according to standard methods (Lichter et al., 1988).
  • a digoxigenin-labeled centromere-specific ⁇ -satellite probe To identify chromosome 7, a digoxigenin-labeled centromere-specific ⁇ -satellite probe (Oncor) was co-hybridized and detected with antidigoxigenin-FITC. Chromosomes were counterstained with DAP1 and visualized directly on the photomicroscope.
  • Genomic DNA samples were amplified by PCR and used in SSCP analyses as described (Orita et al., 1989; Ptacek et al., 1991). Primer pairs used for this study are shown in Table 3. Annealing temperature was 58° C. for all PCR reactions. Reactions (10 ⁇ l) were diluted with 40 ⁇ l of 0.1% SDS/1 mM EDTA and 30 ⁇ l of 95% formamide dye. Diluted products were denatured by heating at 94° C. or 100° C. for 5 or 10 minutes, and 3-5 ⁇ l of each sample were separated by electrophoresis on either 7.5% or 10% non-denaturing polyacrylamide gels (50 acrylamide: 1 Bis-acrylamide) at 4° C. Electrophoresis was carried out at 40-50 watts for 2 to 5 hours. Gels were transferred to 3 MM filter paper, dried and exposed to X-ray film at ⁇ 80° C. for 12-36 hours.
  • Sense-strand oligonucleotides are indicated with an “L” and anti-sense oligonucleotides are indicated with an “R”.
  • cDNA sequence was obtained from the Genbank database, nucleotide numbering begins with the initiator methionine.
  • the phrases “INTRON I”, “INTRON II” and “INTRON III” are from Curran et al. (1995) and correspond to introns 6, 7 and 9, respectively.
  • SSCP conformers were cut directly from dried gels and eluted in 75-100 ⁇ l of distilled water at either 37° C. or 65° C. for 30 minutes. Ten ⁇ l of the eluted DNA was used as template for a second PCR reaction using the original primer pair. Products were fractionated in 2% low-melting temperature agarose gels (FMC), and DNA fragments were purified and sequenced directly by cycle sequencing (Wang and Keating, 1994). Alternatively, purified PCR products were cloned into pBluescript II SK + (Stratagene) using the T-vector method as described (Marchuk et al., 1990). Plasmid DNA samples were purified and sequenced by the dideoxy chain termination method using SequiTherm Polymerase (Epicentre Technologies) or as previously described (Curran et al., 1993a).
  • Exon/intron boundaries were determined by sequencing the cosmids with primers designed to the cDNA. Sequencing revealed the presence of 15 exons (FIG. 8) with sizes ranging from 100 bp (exon 11) to 553 bp (exon 15) (see Table 4). Intron donor and acceptor splice sites did not diverge from the invariant GT and AG. A single pair of primers was designed for most exons and two pairs with overlapping products were designed for exons 4, 6 and 7 (Table 5). Due to repetitive DNA sequences in flanking introns, nested PCR was used to amplify exons 1 and 11. This set of primers can be used to screen the entire coding sequence of HERG for mutations.
  • a multiple tissue Northern blot containing ⁇ 2 ⁇ g/lane of poly-A + mRNA was purchased from Clonetech (Human MIN blot 1).
  • a high specific activity >1.5 ⁇ 10 9 cpm/ ⁇ g DNA
  • radiolabeled HERG cDNA fragment containing nucleotides 679-2239 of the coding sequence was prepared by random hexamer priming as described (Feinberg and Vogelstein, 1983). Probe was added to the hybridization solution at final concentration of 5 ⁇ 10 6 cpm/ml.
  • Hybridization was carried out at 42° C. for 24 hours in 20 ml of Quickhyb solution (Stratagene). Final washes were carried out at 65° C. for 30 minutes in a solution of 0.1% SDS/0.1 ⁇ SSC.
  • LQT2 is linked to markers on chromosome 7q35-36.
  • LQT1, LQT2, LQT3 linkage analyses were performed in families with this disorder.
  • Genotype analyses with polymorphic markers linked to the known LQT loci suggested that the disease phenotype in these families was linked to polymorphic markers on chromosome 7q35-36 (FIG. 14 ).
  • Haplotype analyses were consistent with previous studies, placing LQT2 between D7S505 and D7S483 (FIG. 14; Wang et al., 1995), localizing this gene to chromosome 7q35-36.
  • HERG maps to chromosome 7q35-36.
  • HERG was previously mapped to chromosome 7 (Wannke and Ganetzky, 1994). To test the candidacy of this gene, the localization of HERG was refined using two physical mapping techniques. First, HERG was mapped on a set of yeast artificial chromosome (YAC) contigs constructed for chromosome 7 (Green et al., 1994). HERG was localized to the same YAC as D7S505, a polymorphic marker that was tightly linked to LQT2 (Table 6). Second, HERG was mapped to chromosome 7q35-36 using fluorescent in situ hybridization (FISH) with a P1 genomic clone containing HERG.
  • FISH fluorescent in situ hybridization
  • SSCP analyses were used to identify polymorphisms within HERG, and linkage analyses were performed in the chromosome 7-linked families.
  • Two aberrant SSCP conformers were identified in DNA samples from patients and controls using primer pairs 5-11, and 3-8. These conformers were cloned and sequenced.
  • HERG is LQT2
  • SSCP analyses were used to screen for mutations in affected individuals. Since the genomic structure of HERG was unknown (this portion of the work being performed prior to determining the complete intron/exon structure for the gene), oligonucleotide primer pairs were designed from published (Warmke and Ganetzky, 1994) HERG cDNA sequences (Table 3). In most cases, single products of expected size were generated. For primer pairs 1-10, 6-13, and 15-17, however, products of greater than expected size were obtained, suggesting the presence of intronic sequences. To examine this possibility, these larger products were cloned and sequenced.
  • DNA sequence analyses identified three introns at positions 1557/1558, 1945/1946, and 2398/2399 of the cDNA sequence SEQ ID NO:1 (FIG. 15 ). These boundaries were confirmed by direct DNA sequencing of HERG genomic clones containing HERG (data not shown). To facilitate SSCP analyses, additional primers were designed to intronic sequences.
  • SSCP analyses using primer pair 3-8 identified an A to G polymorphism within HERG (cDNA nucleotide 1692).
  • Analysis of kindred 2287 (K2287) using this SSCP polymorphism defined a pattern of genotypes consistent with a null allele (FIG. 13 ).
  • Possible explanations for these findings included multiple misinheritances, a possibility not supported by previous genotypic analyses, DNA sample errors, base-pair substitutions, or a deletion.
  • PCR analyses of K2287 were repeated using a new primer pair (3-9) flanking the previous set of primers.
  • This mutation results in substitution of valine for a highly conserved alanine at codon 561 (A561V), altering the fifth membrane spanning domain (S5) of the HERG protein (FIG. 12 B).
  • A561V the fifth membrane spanning domain
  • S5 the fifth membrane spanning domain
  • an A to G substitution was identified at position 1408.
  • This mutation results in substitution of aspartic acid for a conserved asparagine at codon 470 (N470D), located in the second membrane spanning domain (S2; FIG. 12 D).
  • N470D conserved asparagine at codon 470
  • S2 second membrane spanning domain
  • K2015 a G to C substitution was identified. This substitution disrupts the splice-donor sequence of intron III (intron 9), affecting the cyclic nucleotide binding domain (FIG. 12 F).
  • K1663 has a G1714T mutation resulting in G572C
  • K2548 has an A1762G mutation resulting in N588D
  • K2554 and K1697 both have a C1841T mutation yielding A614V
  • K1789 has a T1889C mutation resulting in V630A. None of the aberrant conformers was identified in DNA samples from more than 200 unaffected individuals.
  • SSCP was used to screen for mutations in sporadic cases.
  • Primer pair 4-12 identified an aberrant conformer in affected individual II-1 of K2269 (FIG. 14 A). This conformer was not identified in either parent or in more than 200 unaffected individuals.
  • Direct DNA sequencing of the aberrant conformer identified a G to A substitution at position 1882. This mutation results in substitution of serine for a highly conserved glycine at codon 628 (G628S) (FIG. 14 B), altering the pore forming domain. Genotype analysis of this kindred using nine informative STR polymorphisms confirmed maternity and paternity.
  • HERG is expressed in the heart.
  • HERG was originally identified from a hippocampal cDNA library (Warmke and Ganetzky, 1994). To determine the tissue distribution of HERG mRNA, partial cDNA clones were isolated and used in Northern analyses. Northern analyses showed strongest hybridization to heart mRNAs, with faint signals in brain, liver, and pancreas (FIG. 15 ). Non-specific hybridization was also seen in lung, possibly due to genomic DNA contamination. The size of the bands observed in cardiac mRNA was consistent with the predicted size of HERG. Two bands, of ⁇ 4.1 and 4.4 kb were identified, possibly due to alternative splicing or the presence of a second related mRNA. These data indicate that HERG is strongly expressed in the heart, consistent with its involvement in LQT.
  • the mutations that were identified are consistent with a dominant-negative mechanism. Two mutations result in premature stop codons and truncated proteins ( ⁇ 1261 and the splice-donor mutation). In the first case, only the amino terminus and a portion of the first membrane spanning domain (S1) remain. In the second, the carboxyl end of the protein is truncated, leaving all membrane spanning domains intact. HERG contains a cyclic nucleotide binding domain near the carboxyl terminus, and in both mutations this domain is deleted. In another mutation, an in-frame deletion of nine amino acids disrupts the third membrane spanning domain ( ⁇ I500-F508).
  • missense mutations also affect membrane spanning domains, A561V in the S5 domain and N470D in S2. Both mutations affect amino acids conserved in the eag family of potassium channels and likely alter the protein's secondary structure.
  • the de novo missense mutation, G628S occurs in the pore-forming domain. This domain is highly conserved in all potassium channel ⁇ -subunits. This mutation affects a conserved amino acid that is of known importance for ion selectivity. When this substitution was introduced into Shaker H4, potassium ion selectivity was lost (Heginbotham et al., 1994). As discussed above, these mutations could induce the loss of HERG function.
  • LQT-related arrhythmias suggests that mutations in cardiac-specific ion channel genes, or genes that modulate cardiac ion channels, cause delayed myocellular repolarization. Delayed myocellular repolarization could promote reactivation of L-type calcium channels, resulting in secondary depolarizations (January and Riddle, 1989). These secondary depolarizations are the likely cellular mechanism of torsade de pointes arrhythmias (Surawicz, 1989). This hypothesis is supported by the observation that pharmacologic block of potassium channels can induce QT prolongation and repolarization-related arrhythmias in humans and animal models (Antzelevitch and Sicouri, 1994). The discovery that one form of LQT results from mutations in a cardiac potassium channel gene supports the myocellular hypothesis.
  • ⁇ -adrenergic receptor activation increases intracellular cAMP and enhances L-type Ca 2+ channel function. Cyclic AMP may also activate HERG, thereby increasing net outward current and accelerating the rate of myocellular repolarization. Dominant-negative mutations of HERG might interrupt the normal modulation of HERG function by cAMP, thereby permitting a predominant effect on L-type Ca 2+ channel function. The resulting imbalance would increase the likelihood that enhanced sympathetic tone could induce Ca 2+ channel-dependent secondary depolarizations, the probable cellular mechanism of torsade de pointes. ⁇ -adrenergic blocking agents could act by interrupting the effect of cAMP on L-type Ca 2+ channels, possibly explaining the beneficial effects of ⁇ -blockers in some LQT patients.
  • Segments of HERG coding sequence are expressed as fusion protein in E. coli.
  • the overexpressed protein is purified by gel elution and used to immunize rabbits and mice using a procedure similar to the one described by Harlow and Lane, 1988. This procedure has been shown to generate Abs against various other proteins (for example, see Kraemer et al., 1993).
  • HERG coding sequence is cloned as a fusion protein in plasmid PET5A (Novagen, Inc., Madison, Wis.). After induction with IPTG, the overexpression of a fusion protein with the expected molecular weight is verified by SDS/PAGE. Fusion protein is purified from the gel by electroelution. Identification of the protein as the HERG fusion product is verified by protein sequencing at the N-terminus. Next, the purified protein is used as immunogen in rabbits.
  • Rabbits are immunized with 100 ⁇ g of the protein in complete Freund's adjuvant and boosted twice in 3 week intervals, first with 100 ⁇ g of immunogen in incomplete Freund's adjuvant followed by 100 ⁇ g of immunogen in PBS. Antibody containing serum is collected two weeks thereafter.
  • Monoclonal antibodies are generated according to the following protocol. Mice are immunized with immunogen comprising intact HERG or HERG peptides (wild type or mutant) conjugated to keyhole limpet hemocyanin using glutaraldehyde or EDC as is well known.
  • the immunogen is mixed with an adjuvant.
  • Each mouse receives four injections of 10 to 100 ⁇ g of immunogen and after the fourth injection blood samples are taken from the mice to determine if the serum contains antibody to the immunogen.
  • Serum titer is determined by ELISA or RIA. Mice with sera indicating the presence of antibody to the immunogen are selected for hybridoma production.
  • Spleens are removed from immune mice and a single cell suspension is prepared (see Harlow and Lane, 1988). Cell fusions are performed essentially as described by Kohler and Milstein, 1975. Briefly, P3.65.3 myeloma cells (American Type Culture Collection, Rockville, Md.) are fused with immune spleen cells using polyethylene glycol as described by Harlow and Lane, 1988. Cells are plated at a density of 2 ⁇ 10 5 cells/well in 96 well tissue culture plates. Individual wells are examined for growth and the supernatants of wells with growth are tested for the presence of HERG specific antibodies by ELISA or RIA using wild type or mutant HERG target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonality.
  • Clones with the desired specificities are expanded and grown as ascites in mice or in a hollow fiber system to produce sufficient quantities of antibody for characterization and assay development.
  • Monoclonal antibody is attached to a solid surface such as a plate, tube, bead, or particle.
  • the antibody is attached to the well surface of a 96-well ELISA plate.
  • 100 ⁇ l sample e.g., serum, urine, tissue cytosol
  • the sample is incubated for 2 hrs at room temperature. Next the sample fluid is decanted, and the solid phase is washed with buffer to remove unbound material.
  • 100 ⁇ l of a second monoclonal antibody to a different determinant on the HERG peptide/protein is added to the solid phase.
  • This antibody is labeled with a detector molecule (e.g., 125 I, enzyme, fluorophore, or a chromophore) and the solid phase with the second antibody is incubated for two hrs at room temperature. The second antibody is decanted and the solid phase is washed with buffer to remove unbound material.
  • a detector molecule e.g., 125 I, enzyme, fluorophore, or a chromophore
  • the amount of bound label which is proportional to the amount of HERG peptide/protein present in the sample, is quantified. Separate assays are performed using monoclonal antibodies which are specific for the wild-type HERG as well as monoclonal antibodies specific for each of the mutations identified in HERG.
  • the HERG cDNA was subcloned into a poly-A + expression vector and the 5′ and 3′ UTRs reduced to minimal lengths.
  • the final HERG expression construct contains cDNA sequence from nucleotides ⁇ 6 through 3513 in the pSP64 plasmid vector (Promega).
  • the HERG construct was characterized by restriction mapping and DNA sequence analyses.
  • Complementary RNAs for injection into oocytes were prepared with the mCAP RNA Capping Kit (Stratagene) following linearization of the expression construct with EcoRI.
  • Xenopus frogs were anesthetized by immersion in 0.2% tricaine for 15-30 min. Ovarian lobes were digested with 2 mg/ml Type 1A collagenase (Sigma) in Ca 2+ -free ND96 solution for 1.5 hours to remove follicle cells. Stage IV and V oocytes (Dumont, 1972) were injected with HERG cRNA (0.05 mg/ml, 50 nl), then cultured in Barth's solution supplemented with 50 ⁇ g/ml gentamycin and 1 mM pyruvate at 18° C.
  • HERG cRNA 0.05 mg/ml, 50 nl
  • Barth's solution contained (in mM): 88 NaCl, 1 KCl, 0.4 CaCl 2 , 0.33 Ca(NO 3 ) 2 , 1 MgSO 4 , 2.4 NaHCO 3 , 10 HEPES; pH 7.4.
  • oocytes were bathed in ND96 solution. This solution contained (in mM): 96 NaCl, 2 KCl, 1 MgCl 2 , 1.8 CaCl 2 , 5 HEPES; pH 7.6. In some experiments, KCl was varied by equimolar substitution with NaCl. Currents were recorded at room temperature (21-23° C.) using standard two-microelectrode voltage clamp techniques. Glass microelectrodes were filled with 3 M KCl and their tips broken to obtain tip resistances of 1-3 M ⁇ for the voltage-recording electrode and 0.6-1 M ⁇ for the current-passing electrode. Oocytes were voltage-clamped with a Dagan TEV-200 amplifier.
  • Voltage commands were generated using pClamp software (ver. 6, Axon Instruments), a 486DX2 personal computer and a TL-1 D/A interface (Axon Instruments).
  • Current signals were digitally sampled at a rate equal to 2-4 times the low-pass cut-off frequency ( ⁇ 3 db) of a 4-pole Bessel filter. Unless indicated, currents were corrected for leak and capacitance using standard, on-line P/3 leak subtraction.
  • the oocyte membrane potential was held at ⁇ 70 mV between test pulses, applied at a rate of 1-3 pulses/min. Data analyses, including exponential fitting of current traces, were performed using pCLAMP.

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